Process for the synthesis of oligomeric compounds

ABSTRACT

Synthetic processes are provided wherein oligomeric compounds are prepared having phosphodiester, phosphorothioate, phosphorodithioate, or other covalent linkages. Also provided are synthetic intermediates useful in such processes.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation in part of U.S. Ser. No.09/111,678, filed Jul. 8, 1998, the content of which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

[0002] This invention relates to methods for the preparation ofoligomeric compounds having phosphite, phosphodiester, phosphorothioate,phosphorodithioate or other linkages, and to intermediates useful intheir preparation.

BACKGROUND OF THE INVENTION

[0003] Oligonucleotides and their analogs have been developed and usedin molecular biology in a variety of procedures as probes, primers,linkers, adapters, and gene fragments. Modifications to oligonucleotidesused in these procedures include labeling with nonisotopic labels, e.g.fluorescein, biotin, digoxigenin, alkaline phosphatase, or otherreporter molecules. Other modifications have been made to the ribosephosphate backbone to increase the nuclease stability of the resultinganalog. Examples of such modifications include incorporation of methylphosphonate, phosphorothioate, or phosphorodithioate linkages, and2′-O-methyl ribose sugar units. Further modifications include those madeto modulate uptake and cellular distribution. With the success of thesecompounds for both diagnostic and therapeutic uses, there exists anongoing demand for improved oligonucleotides and their analogs.

[0004] It is well known that most of the bodily states in multicellularorganisms, including most disease states, are effected by proteins. Suchproteins, either acting directly or through their enzymatic or otherfunctions, contribute in major proportion to many diseases andregulatory functions in animals and man. For disease states, classicaltherapeutics has generally focused upon interactions with such proteinsin efforts to moderate their disease-causing or disease-potentiatingfunctions. In newer therapeutic approaches, modulation of the actualproduction of such proteins is desired. By interfering with theproduction of proteins, the maximum therapeutic effect may be obtainedwith minimal side effects. It is therefore a general object of suchtherapeutic approaches to interfere with or otherwise modulate geneexpression, which would lead to undesired protein formation.

[0005] One method for inhibiting specific gene expression is with theuse of oligonucleotides, especially oligonucleotides which arecomplementary to a specific target messenger RNA (mRNA) sequence.Several oligonucleotides are currently undergoing clinical trials forsuch use. Phosphorothioate oligonucleotides are presently being used assuch antisense agents in human clinical trials for various diseasestates, including use as antiviral agents.

[0006] Transcription factors interact with double-stranded DNA duringregulation of transcription. Oligonucleotides can serve as competitiveinhibitors of transcription factors to modulate their action. Severalrecent reports describe such interactions (see Bielinska, A., et. al.,Science, 1990, 250, 997-1000; and Wu, H., et. al., Gene, 1990, 89,203-209).

[0007] In addition to such use as both indirect and direct regulators ofproteins, oligonucleotides and their analogs also have found use indiagnostic tests. Such diagnostic tests can be performed usingbiological fluids, tissues, intact cells or isolated cellularcomponents. As with gene expression inhibition, diagnostic applicationsutilize the ability of oligonucleotides and their analogs to hybridizewith a complementary strand of nucleic acid. Hybridization is thesequence specific hydrogen bonding of oligomeric compounds viaWatson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases ofsuch base pairs are said to be complementary to one another.

[0008] Oligonucleotides and their analogs are also widely used asresearch reagents. They are useful for understanding the function ofmany other biological molecules as well as in the preparation of otherbiological molecules. For example, the use of oligonucleotides and theiranalogs as primers in PCR reactions has given rise to an expandingcommercial industry. PCR has become a mainstay of commercial andresearch laboratories, and applications of PCR have multiplied. Forexample, PCR technology now finds use in the fieldse of forensics,paleontology, evolutionary studies and genetic counseling.Commercialization has led to the development of kits which assistnon-molecular biology-trained personnel in applying PCR.Oligonucleotides and their analogs, both natural and synthetic, areemployed as primers in such PCR technology.

[0009] Oligonucleotides and their analogs are also used in otherlaboratory procedures. Several of these uses are described in commonlaboratory manuals such as Molecular Cloning, A Laboratory Manual,Second Ed., J. Sambrook, et al., Eds., Cold Spring Harb:or LaboratoryPress, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel,et al., Eds., Current Publications, 1993. Such uses include as syntheticoligonucleotide probes, in screening expression libraries withantibodies and oligomeric compounds, DNA sequencing, in vitroamplification of DNA by the polymerase chain reaction, and insite-directed mutagenesis of cloned DNA. See Book 2 of MolecularCloning, A Laboratory Manual, supra. See also “DNA-protein interactionsand The Polymerase Chain Reaction” in Vol. 2 of Current Protocols InMolecular Biology, supra.

[0010] Oligonucleotides and their analogs can be synthesized to havecustomized properties that can be tailored for desired uses. Thus anumber of chemical modifications have been introduced into oligomericcompounds to increase their usefulness in diagnostics, as researchreagents and as therapeutic entities. Such modifications include thosedesigned to increase binding to a target strand (i.e. increase theirmelting temperatures, Tm), to assist in identification of theoligonucleotide or an oligonucleotide-target complex, to increase cellpenetration, to stabilize against nucleases and other enzymes thatdegrade or interfere with the structure or activity of theoligonucleotides and their analogs, to provide a mode of disruption(terminating event) once sequence-specifically bound to a target, and toimprove the pharmacokinetic properties of the oligonucleotide.

[0011] The chemical literature discloses numerous processes for couplingnucleosides through phosphorous-containing covalent linkages to produceoligonucleotides of defined sequence. One of the most popular processesis the phosphoramidite technique (see, e.g., Advances in the Synthesisof Oligonucleotides by the Phosphoramidite Approach, Beaucage, S. L.;Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311 and references citedtherein), wherein a nucleoside or oligonucleotide having a free hydroxylgroup is reacted with a protected cyanoethyl phosphoramidite monomer inthe presence of a weak acid to form a phosph.te-linked structure.Oxidation of the phosphite linkage followed by hydrolysis of thecyanoethyl group yields the desired phosphodiester or phosphorothioatelinkage.

[0012] The phosphoramidite technique, however, has significantdisadvantages. For example, cyanoethyl phosphoramidite monomers arequite expensive. Although considerable quantities of monomer gounreacted in a typical phosphoramidite coupling, unreacted monomer canbe recovered, if at all, only with great difficulty.

[0013] Another disadvantage of using a β-eliminating cyanoethoxy groupis formation of acrylonitrile upon removal of the phosphorus protectinggroup. Acrylonitrile is a highly toxic agent as well as a suspectedcarcinogen (See 1994-1995 Aldrich Chemical Company Catalog, at page 32).Acrylonitrile is also suspected of forming cyclic structures withthymidine resulting in oligomeric compounds having decreasedhybridization ability. These modified oligomeric compounds areundesirable because they are difficult to separate from the desiredoligomeric compound.

[0014] Consequently, there remains a need in the art for syntheticmethods that will overcome these problems.

[0015] Several processes are known for the solid phase synthesis ofoligonucleotide compounds. These are generally disclosed in thefollowing U.S. Patents: U.S. Pat. No. 4,458,066; issued Jul. 3, 1984;U.S. Pat. No. 4,500,707, issued Feb. 19, 1985; and U.S. Pat. No.5,132,418, issued Jul. 21, 1992. Additionally, a process for thepreparation of oligonucleotides using phosphoramidite intermediates isdisclosed in U.S. Pat. No. 4,973,679, issued Nov. 27, 1990.

[0016] A process for the preparation of phosphoramidites is disclosed inU.S. Pat. No. 4,415,732, issued Nov. 15, 1983.

[0017] Phosphoramidite nucleoside compounds are disclosed in U.S. Pat.No. 4,668,777, issued May 26, 1987.

[0018] A process for the preparation of oligonucleotides using aβ-eliminating phosphorus protecting group is disclosed in U.S. Pat. No.Re. 34,069, issued Sep. 15, 1992.

[0019] A process for the preparation of oligonucleotides using aβ-eliminating or allylic phosphorus protecting group is disclosed inU.S. Pat. No. 5,026,838, issued Jun. 25, 1991.

SUMMARY OF THE INVENTION

[0020] In one aspect of the present invention, methods are provided forthe preparation of oligomeric compounds comprising a moiety having theFormula I:

[0021] wherein:

[0022] A is a monocyclic or bicyclic aromatic ring system;

[0023] R¹¹ and R₁₂ are each independently H, alkyl, aryl, heteroalkyl,heteroaryl, alkaryl, or aralkyl;

[0024] or R₁₁ and R₁₂ together with the carbon atoms to which they areattached form an optionally substituted aliphatic or aromatic ringhaving from 4 to 6 ring atoms;

[0025] X₄ is alkaryl, aralkyl, sulfoxyl, sulfonyl, thio, substitutedsulfoxyl, substituted sulfonyl, or substituted thio, wherein saidsubstituent is alkyl, aryl, or alkaryl;

[0026] or X₄ is a group of formula —C (═O)—(O) _(aa)—R₄₀ where aa is 0or 1 and R₄₀ is lower alkyl, aryl, aralkyl, heteroaryl wherein saidlower alkyl, aryl, aralkyl or heteroaryl groups are optionallysubstituted with one or more alkyl, aryl, aralkyl, halo or acetylgroups;

[0027] or X₄ is a group of formula —(—CH₂—CH₂—)_(d)Si(R₉) ₃ where d is 0or 1;

[0028] each R₉ is, independently, alkyl having 1 to about 10 carbonatoms, or aryl having 6 to about 10 carbon atoms;

[0029] X₁ and X₅ are each independently O or S; comprising:

[0030] (a) providing a compound having the Formula II:

[0031] wherein:

[0032] each R₁, is, independently, H, hydroxyl, C₁-C₂₀ alkyl, C₃-C₂₀alkenyl, C₂-C₂₀ alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso,nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, or polyether;

[0033] or R₁ is a group of formula Z—R₂₂—(R₂₃)_(v);

[0034] Z is O, S, NH, or N—R₂₂—(R₂₃)_(v);

[0035] R₂₂ is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl;

[0036] R₂₃ is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl,nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl,S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl,S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido,hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide,disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reportermolecule, conjugate, polyamine, polyamide, polyalkylene glycol,polyether, a group that enhances the pharmacodynamic properties ofoligonucleotides, or a group that enhances the pharmacokineticproperties of oligonucleotides;

[0037] v is from 0 to about 10;

[0038] or R₁ has the formula:

[0039] y1 is 0 or 1;

[0040] y2 is independently 0 to 10;

[0041] y3 is 1 to 10;

[0042] E is C₁-C₁₀ alkyl, N(Q₁) (Q₂) or N═C(Q₁) (Q₂)

[0043] each Q₁ and Q₂ is, independently, H, C₁-C₁₀ alkyl, substitutedC₁-C₁₀ alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tetheredor untethered conjugate group, a linker to a solid support; or Q₁ andQ₂, together, are joined in a nitrogen protecting group or a ringstructure that can include at least one additional heteroatom selectedfrom N and O;

[0044] or R₁ has one of formula XI or XII:

[0045]  wherein

[0046] Z₀ is O, S, or NH;

[0047] q¹ is from 0 to 10;

[0048] q² is from 1 to 10;

[0049] q³ is 0 or 1;

[0050] q⁴ is, 0, 1 or 2;

[0051] Z₄ is OM₁, SM₁, or N(M₁)₂;

[0052] each M₁ is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,C(═NH)N(H)M₂, C(═O)N(H)M₂ or OC(═O)N(H)M₂;

[0053] M₂ is H or C₁-C₈ alkyl;

[0054] Z₁, Z₂ and Z₃ comprise a ring system having from about 4 to about7 carbon atoms, or having from about 3 to about 6 carbon atoms and 1 or2 hetero atoms wherein said hetero atoms are selected from oxygen,nitrogen and sulfur, and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic; and

[0055] Z₅ is alkyl or haloalkyl having 1 to about 10 carbon atoms,alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10carbon atoms, aryl having e to about 14 carbon atoms, N(Q₁) (Q₂), OQ₁,halo, SQ₁ or CN;

[0056] R₃ is hydrogen, a hydroxyl protecting group, or a linkerconnected to a solid support;

[0057] each B, independently, is a naturally occurring or non-naturallyoccurring nucleobase or a protected naturally occurring or non-naturallyoccurring nucleobase;

[0058] n is 0 to about 50;

[0059] M is an optionally protected internucleoside linkage;

[0060] R₅ is —N (R₆) ₂, or a heterocycloalkyl or heterocycloalkenyl ringcontaining from 4 to 7 atoms, and having up to 3 heteroatoms selectedfrom nitrogen, sulfur, and oxygen; and

[0061] R₆ is straight or branched chain alkyl having from 1 to 10carbons; and

[0062] (b) reacting the compound of Formula II with a compound havingFormula III:

[0063] wherein m is 0 to about 50;

[0064] R_(3a) is hydrogen;

[0065] R₂ is hydrogen, a hydroxyl protecting group, or a linkerconnected to a solid support, provided that R₂ and R_(3a) are not bothsimultaneously a linker connected to a solid support; to form theoligomeric compound.

[0066] Some preferred embodiments of the methods of the inventionfurther comprise the step of oxidizing or sulfurizing the oligomericcompound. In some preferred embodiments, the methods of the inventionfurther comprise transforming the oxidized or sulfurized oligomericcompound to form a further compound having the Formula III, where m isincreased by 1. Other prefered embodiments further comprise a cappingstep, performed prior to or subsequent to oxidation or sulfurization.

[0067] In some preferred embodiments, the methods of the inventionfurther comprising the step of cleaving the oligomeric compound from thesolid support to produce a compound having the Formula IV:

[0068] In some preferred embodiments of the methods of the invention, Ais phenyl or a naphthalene.

[0069] In further preferred embodiments of the methods of the invention,X₄ is benzoyl, acetyl (—C(═O)—CH₃) or levulinyl.

[0070] In some partiularly preferred embodiments, X₄ is benzoyl, acetylor levulinyl, A is phenyl, with the moiety —OX₄ being in the ortho orpara position thereof, with the ortho position being more preferred.

[0071] In further preferred embodiments of the methods of the invention,X₄ is benzoyl, acetyl or levulinyl, A is a naphthalene ring connected toX₅ at the 1-position, with the moiety —OX₄ being in the 5- or 6-positionof the naphthalene ring.

[0072] In further preferred embodiments of the invention, each R₆ isisopropyl.

[0073] In some especially preferred embodiments of the invention, n is0. In further prefered embodiments, at least one of X₁ and X₅ is 0. Morepreferably, X₁ and X₅ are each O.

[0074] In some especially preferred emboiments, n is 0; X₄ is benzoyl,acetyl or levulinyl; A is phenyl; —OX₄ is in the ortho or para position,with the ortho position being more preferred; X₁ and X₅ are each O; andR₅ is diisopropylamino.

[0075] In some preferred embodiments, the compound of Formula II isobtained by reaction of a compound having Formula V:

[0076] with a compound having the Formula VI:

[0077] in the presence of an acid, pref erably tetrazole. Preferably, R₅is N,N-diisopropylamino.

[0078] In other preferred embodiments, the compound of Formula II isobtained by (a) reacting a compound having Formula-V with achlorophosphine compund of formula ClP(R₅)₂ in the presence of a base;and

[0079] (b) contacting the product of step (a) with a compound of FormulaXX:

[0080] in the presence of an acid. Preferably, the chlorophosphinecompound has the formula ClP[(i-Pr)₂N]₂.

[0081] Also provided in accordance with the present invention arecompounds having Formula VII:

[0082] wherein:

[0083] A is a monocyclic or bicyclic aromatic ring system;

[0084] R₁₁ and R₁₂ are each independently H, alkyl, aryl, heteroaryl,alkaryl, or aralkyl;

[0085] or R₁₁ and R₁₂ together with the carbon atoms to which they areattached form an optionally substituted aliphatic or aromatic ringhaving from 4 to 6 ring atoms;

[0086] X₄ is alkaryl, aralkyl, sulfoxyl, sulfonyl, thio, substitutedsulfoxyl, substituted sulfonyl, or substituted thio, wherein saidsubstituent is alkyl, aryl, or alkaryl;

[0087] or X₄ is a group of formula —C (═O)—(O)_(aa)—R₄₀ where aa is 0 or1 and R₄₀ is lower alkyl, aryl, aralkyl, heteroaryl wherein said loweralkyl, aryl, aralkyl or heteroaryl groups are optionally substitutedwith one or more alkyl, aryl, aralkyl, halo or acetyl groups;

[0088] or X₄ is a group of formula —(—CH₂—CH₂—)_(d)Si(R₉)₃ where d is 0or 1;

[0089] each R₉ is, independently, alkyl having 1 to about 10 carbonatoms, or aryl having 6 to about 10 carbon atoms;

[0090] X₁ and X₅ are each independently O or S;

[0091] D is (R₇) (R₈) P—or (R₇) (R₈) P (═X₂)—;

[0092] R₈ is R₅, or has the Formula VIII:

[0093]  wherein:

[0094] each R₁, is, independently, H, hydroxyl, C₁-C₂₀ alkyl, C₃-C₂₀alkenyl, C₂-C₂₀ alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso,nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, or polyether;

[0095] or R₁ is a group of formula Z—R₂₂—(R₂₃)_(v);

[0096] Z is O, S, NH, or N—R₂₂—(R₂₃)_(v);

[0097] R₂₂ is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl;

[0098] R₂₃ is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl,nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl,S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl,S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido,hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide,disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reportermolecule, conjugate, polyamine, polyamide, polyalkylene glycol,polyether, a group that enhances the pharmacodynamic properties ofoligonucleotides, or a group that enhances the pharmacokineticproperties of oligonucleotides;

[0099] v is from 0 to about 10;

[0100] or R₁ has the formula:

[0101] y1 is 0 or 1;

[0102] y2 is independently 0 to 10;

[0103] y3 is 1 to 10;

[0104] E is C₁-C₁₀ alkyl, N(Q₁) (Q₂) or N═C(Q₁) (Q₂);

[0105] each Q₁ and Q₂ is, independently, H, C₁-C₁₀ alkyl, substitutedC₁-C₁₀ alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tetheredor untethered conjugate group, a linker to a solid support; or Q₁ andQ₂, together, are joined in a nitrogen protecting group or a ringstructure that can include at least one additional heteroatom selectedfrom N and O;

[0106] or R₁ has one of formula XI or XII:

[0107]  wherein

[0108] Z⁰ is O, S, or NH;

[0109] q¹ is from 0 to 10;

[0110] q² is from 1 to 10;

[0111] q³ is 0 or 1;

[0112] q⁴ is, 0, 1 or 2;

[0113] Z₄ is OM₁, SM₁, or N(M₁) ₂;

[0114] each M₁ is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,C(═NH)N(H)M₂, C(═O)N(H)M₂ or OC(═O)N(H)M₂;

[0115] M₂ is H or C₁-C₈ alkyl;

[0116] Z₁, Z₂ and Z₃ comprise a ring system having from about 4 to about7 carbon atoms, or having from about 3 to about 6 carbon atoms and 1 or2 hetero atoms wherein said hetero atoms are selected from oxygen,nitrogen and sulfur, and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic; and

[0117] Z₅ is alkyl or haloalkyl having 1 to about 10 carbon atoms,alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q₁) (Q₂), OQ₁,halo, SQ₁ or CN;

[0118] each X₂ is O or S;

[0119] R₅ is —N(R₆)₂, or a heterocycloalkyl for heterocycloalkenyl ringcontaining from 4 to 7 atoms, and having up to 3 heteroatoms selectedfrom nitrogen, sulfur, and oxygen;

[0120] M is an optionally protected internucleoside linkage;

[0121] m is 0 to about 50;

[0122] each B, independently, is a naturally occurring or non-naturallyoccurring nucleobase or a protected naturally occurring or non-naturallyoccurring nucleobase; and

[0123] R₇ is R₅, or has the Formula IX:

[0124]  wherein:

[0125] R₃ is hydrogen, a hydroxyl protecting group, or a linkerconnected to a solid support;

[0126] n is 0 to about 50; with the proviso that the sum of m and n donot exceed 50.

[0127] In some preferred embodiments, X₄ is benzoyl, acetyl orlevulinyl, with acetyl being preferred.

[0128] In some partiularly preferred embodiments, X₄ is benzoyl, acetylor levulinyl, A is phenyl, with the moiety —OX₄ being in the ortho orpara position, with the ortho position being more preferred, and R₁₁ andR₁₂ are each H.

[0129] In further preferred embodiments of the methods of the invention,X₄ is benzoyl, acetyl or levulinyl, A is a naphthalene ring connected toX₅ at the 1-position, with the moiety —OX₄ being at the 5- or 6-positionof the naphthalene ring, and R₁₁ and R₁₂ are each H.

[0130] In some preferred embodiments, at least one of X₁ and X₅ is O. Inmore preferred embodiments, X₁ and X₅ are each O.

[0131] In particularly preferred embodiments, X₄ is benzoyl, acetyl orlevulinyl, A is phenyl with —OX₄ being in the ortho or para position, X₁and X₅ are each O, and R₁₁ and R₁₂ are each H.

[0132] In some preferred embodiments, R₈ is R₅. In further preferredembodiments, n is 0. In still further preferred embodiments, R₈ is R₅, nis 0, X₄ is benzoyl, acetyl or levulinyl, A is phenyl with —OX₄ attachedat the ortho or para position; X₁ and X₅ are each O, and R₁₁ and R₁₂ areeach H.

[0133] In some preferred embodiments, R₈ has the Formula VIII, and R₇has the Formula IX. In further preferred embodiments, R₈ has the FormulaVIII, and R₇ has the Formula IX, and n is 0. In still further preferredembodiments, R₈ has the Formula VIII, and R₇ has the Formula IX, and nis 0 and m is 0. In still further preferred embodiments, R₈ has theFormula VIII, and R₇ has the Formula IX, n is 0, X₄ is benzoyl, acetylor levulinyl, A is phenyl with —OX₄ attached at the ortho or paraposition, X₁ and X₅ are each O, R₅ is diisopropylamino, and R₁₁ and R₁₂are each H.

[0134] In some preferred embodiments, at least one of X₁ and X₅ is S. Infurther preferred embodiments, A is (R₇) (R₈)P—.

[0135] In some preferred embodiments, the present invention providescompounds comprising a moiety of Formula:

[0136] wherein the constituent variables are as previously defined.

[0137] Preferably, the moiety A is phenyl with —OX₄ attached at theortho or para position, with the ortho position being preferred; or A isnaphthalene connected to X, at the 1-position, and the moiety —OX₄ isattached to the 5- or 6-position of the naphthalene ring. In especiallypreferred embodiments, X₄ is benzoyl, acetyl or levulinyl, A is phenylwith —OX₄ is in the ortho or para position, and X₁ and X₅ are each O.

[0138] The present invention also provides compounds having Formula X:

[0139] wherein m and n are each independently from 0 to about 50,provided that the sum of m and n does not exceed 50; and the otherconstituent variables are as previously defined.

[0140] In some preferred embodiments, R₂ is a linker connected to asolid support.

[0141] Also provided in accordance with the present invention aremethods for the preparation of a compound of Formula II:

[0142] wherein the consitituent variables are as previously defified,comprising:

[0143] (a) selecting a 5′-protected nucleoside having Formula V:

[0144] and

[0145] (b) re acting the nucleoside with a compound having the FormulaVI:

[0146] in the presence of an acid.

[0147] The present invention also provides methods for the preparationof a compound of Formula II comprising:

[0148] (a) selecting a 5′-protected nucleoside of Formula V:

[0149] (b) reacting the protected nucleoside with a chlorophosphinecompund of formula ClP(R₅)₂ in the presence of a base; and

[0150] (c) contacting the product of step (b) with a compound of FormulaXX:

[0151] in the presence of an acid;

[0152] to form the nucleoside phosphoramidite.

[0153] In some preferred embodiments of the compounds of Formulas X andX₁ , m and n are each 0.

[0154] Also provided in accordance with the present invention arecompounds having the formula:

[0155] wherein A, X₁, X₄ X₅, R₁₁ and R₁₂ are as defined above, X₂ ishalogen, and X₃ is —N(R₆) ₂, or a heterocycloalkyl or heterocycloalkenylring containing from 4 to 7 atoms, and having up to 3 heteroatomsselected from nitrogen, sulfur, and oxygen. In further preferredembodiments, A is phenyl with —O—X₄ in the ortho or para position, X₁and X₅ are O, and R₁₁ and R₁₂ are each H, and X₄ is benzoyl, acetyl orlevulinyl. In still further preferred embodiments, X₃ is —N(R₆) ₂ whereR₆ is isopropyl. Preferably, X₂ is chlorine.

[0156] The present invention also provides products produced by themethods of the invention.

DESCRIPTION OF PREFERRED EM(BODIMENTS

[0157] The present invention provides methods for the preparation ofoligomeric compounds having phosphite, phosphodiester, phosphorothioate,or phosphorodithioate linkages, and to intermediates useful in theirpreparation.

[0158] In some preferred embodiments of the invention, methods areprovided for the preparation of an oligomeric compound comprising atleast one moiety having the Formula I:

[0159] wherein:

[0160] A is a monocyclic or bicyclic aromatic ring system;

[0161] R₁₁ and R₁₂ are each independently H, alkyl, aryl, heteroaryl,alkaryl, or aralkyl;

[0162] or R₁₁ and R₁₂ together with the carbon atoms to which they areattached form an optionally substituted aliphatic or aromatic ringhaving from 4 to 6 ring atoms;

[0163] X₄ is alkaryl, aralkyl, sulfoxyl, sulfonyl, thio, substitutedsulfoxyl, substituted sulfonyl, or substituted thio, wherein saidsubstituent is alkyl, aryl, or alkaryl;

[0164] or X₄ is a group of formula —C(═O)—(O)_(aa)—R₄₀ where aa is 0 or1 and R₄₀ is lower alkyl, aryl, aralkyl, heteroaryl wherein said loweralkyl, aryl, aralkyl or heteroaryl groups are optionally substitutedwith one or more alkyl, aryl, aralkyl, halo or acetyl groups;

[0165] or X₄ is a group of formula —(—CH₂—CH₂—)_(d)Si(R₉)₃ where d is 0or 1;

[0166] each R₉ is, independently, alkyl having 1 to about 10 carbonatoms, or aryl having 6 to about 10 zarbon atoms;

[0167] X₁ and X₅ are each independently O or S; comprising:

[0168] (a) providing a compound having the Formula II:

[0169]  wherein:

[0170] each R₁, is, independently, H, hydroxyl, C₁-C₂₀ alkyl, C₃-C₂₀alkenyl, C₂-C₂₀ alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso,nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, or polyether;

[0171] or R₁ is a group of formula Z—R₂₂—(R₂₃)_(v);

[0172] Z is O, S, NH, or N—R₂₂—(R₂₃)_(v);

[0173] R₂₂ is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, Or C₂-C₂₀ alkynyl;

[0174] R₂₃ is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl,nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl,O-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl,S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido,hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide,disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reportermolecule, conjugate, polyamine, polyamide, poly-alkylene glycol,polyether, a group that enhances the pharmaco-dynamic properties ofoligonucleotides, or a group that enhances the pharmacokineticproperties of oligonucleotides;

[0175] v is from 0 to about 10;

[0176] or R₁ has the formula:

[0177] y1 is 0 or 1;

[0178] y2 is independently 0 to 10;

[0179] y3 is 1 to 10;

[0180] E is C₁-C₁₀ alkyl, N(Q₁) (Q₂) or N═C(Q₁) (Q₂);

[0181] each Q₁ and Q₂ is, independently, H, C₁-C₁₀ alkyl, substitutedC₁-C₁₀ alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tetheredor untethered conjugate group, a linker to a solid support; or Q₁ andQ₂, together, are joined in a nitrogen protecting group or a ringstructure that can include at least one additional heteroatom selectedfrom N and O;

[0182] or R₁ has one of formula XI or XIII:

[0183]  wherein

[0184] Z₀ is O, S, or NH;

[0185] q₁ is from 0 to 10;

[0186] q² is from 1 to 10;

[0187] q³ is 0 or 1;

[0188] q⁴ is, 0, 1 or 2;

[0189] Z₄ is OM₁, SM₁, or N(M₁)₂;

[0190] each M₁ is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,C(═NH)N(H)M₂, C(═O)N(H)M₂ or OC(═O)N(H)M₂;

[0191] M₂ is H or C₁-C₈ alkyl;

[0192] Z₁, Z₂ and Z₃ comprise a ring system having from about 4 to about7 carbon atoms, or having from about 3 to about 6 carbon atoms and 1 or2 hetero atoms wherein said hetero atoms are selected from oxygen,nitrogen and sulfur, and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic; and

[0193] Z₅ is alkyl or haloalkyl having 1 to about 10 carbon atoms,alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q₁) (Q₂), OQ₁,halo, SQ₁ or CN;

[0194] R₃ is hydrogen, a hydroxyl protecting group, or a linkerconnected to a solid support;

[0195] each B, independently, is a naturally occurring or non-naturallyoccurring nucleobase or a protected naturally occurring or non-naturallyoccurring nucleobase;

[0196] n is 0 to about 50;

[0197] M is an optionally protected internucleoside linkage;

[0198] R₅ is —N(R₆)₂, or a heterocycloalkyl or heterocycloalkenyl ringcontaining from 4 to 7 atoms, and having up to 3 heteroatoms selectedfrom nitrogen, sulfur, and oxygen; and

[0199] R₆ is straight or branched chain alkyl having from 1 to 10carbons; and

[0200] (b) reacting the compound of Formula II with a compound havingFormula III:

[0201]  wherein m is 0 to about 50;

[0202] R_(3a) is hydrogen;

[0203] R₂ is hydrogen, a hydroxyl protecting group, or a linkerconnected to a solid support, provided that R₂ and R_(3a) are not bothsimultaneously a linker connected to a solid support;

[0204] to form the oligomeric compound.

[0205] The methods of the present invention are useful for thepreparation of oligomeric compounds containing monomeric subunits thatare joined by a variety of linkages, including phosphite,phosphodiester, phosphorothioate, and/or phosphorodithioate linkages. Asused herein, the term “oligomeric compound” is used to refer tocompounds containing a plurality of nucleoside monomer subunits that arejoined by interntcleoside linkages, preferably phosphorus-containinglinkages, such as phosphite, phosphodiester, phosphorothioate, and/orphosphorodithioate linkages. The term “oigomeric compound” thereforeincludes naturally occurring oligonucleotides, their analogs, andsynthetic oligonucleotides. Monomer or higher order synthons havingFormulas II or III above include both native (i.e., naturally occurring)and synthetic (e.g., modified native or totally synthetic) nucleosidesand nucleotides.

[0206] In some preferred embodiments, a phosphoramidite protected at the5′-position is reacted with the 3′-hydroxyl group of a compound ofFormula III to produce phosphite compound containing the linkage ofFormula I. Preferably, capping and/or oxidation or sulfurization stepsare then performed to produce a compound of Formula IV.

[0207] Methods for coupling compounds of Formula II and Formula III ofthe invention include both solution phase and solid phase chemistries.Representative solution phase techniques are described in U.S. Pat. No.5,210,264, which is assigned to the assignee of the present invention.In preferred embodiments, the methods of the present invention areemployed for use in iterative solid phase oligonucleotide syntheticregimes. Representative solid phase techniques are those typicallyemployed for DNA and RNA synthesis utilizing standard phosphoramiditechemistry, (see, e.g., Protocols For Oligonucleotides And Analogs,Agrawal, S., ed., Humana Press, Totowa, N.J., 1993, hereby incorporatedby reference in its entirety). A preferred synthetic solid phasesynthesis utilizes phosphoramidites as activated phosphate compounds. Inthis technique, a phosphoramidite monomer is reacted with a freehydroxyl on the growing oligomer chain to produce an intermediatephosphite compound, which is subsequently oxidized to the p^(v) stateusing standard methods. This technique is commonly used for thesynthesis of several types of linkages including phosphodiester,phosphorothicate, and phosphorodithioate linkages.

[0208] Typically, the first step in such a process is attachment of afirst monomer or higher order subunit containing a protected 5′-hydroxylto a solid support, usually through a linker, using standard methods andprocedures known in the art. See for example, Oligonucleotides AndAnalogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y., 1991,hereby incorporated by reference in its entirety. The support-boundmonomer or higher order first synthon is then treated to remove the5′-protecting group, to form a compound of Formula III wherein R₂ is alinker connected to a solid support. Typically, this is accomplished bytreatment with acid. The solid support bound monomer is then reactedwith a compound of Formula II to form a compound of Formula IV, whichhas a phosphite or thiophosphite linkage of Formula I. In preferredembodiments, synthons of Formula II and Formula III are reacted underanhydrous conditions in the presence of an activating agent such as, forexample, 1H-tetrazole, 5-(4-nitrophenyl)-1H-tetrazole, ordiisopropylamino tetrazolide.

[0209] In some preferred embodiments, phosphite or thiophosphitecompounds containing a linkage of Formula I are oxidized or sulfurizedas shown below to produce compounds having a linkage of Formula XII,where X₁ and X₂ can each be O or S:

[0210] Choice of oxidizing or sulfurizing agent will determine whetherthe linkage of Formula I will be oxidized or sulfurized to aphosphotriester, thiophosphotriester, or a dithiophosphotriesterlinkage.

[0211] It is generally preferable to perform a capping step, eitherprior to or after oxidation or sulfurization of the phosphite triester,thiophosphite triester, or dithiophosphite triester. Such a capping stepis generally known to be beneficial by preventing shortened oligomerchains, by blocking chains that have not reacted in the coupling cycle.One representative reagent used for capping is acetic anhydride. Othersuitable capping reagents and methodologies can be found in U.S. Pat.No. 4,816,571, issued Mar. 28, 1989, hereby incorporated by reference inits entirety.

[0212] Treatment with an acid removes the 5′-hydroxyl protecting group,and thus transforms the solid support bound oligomer into a furthercompound of Formula III wherein R_(3a) is hydrogen, which can thenparticipate in the next synthetic iteration; i.e., which can then bereacted with a further compound of Formula II. This process is repeateduntil an oligomer of desired length is produced.

[0213] The completed oligomer is then cleaved from the solid support.The cleavage step, which can precede or follow deprotection of protectedfunctional groups, will in prefered embodiments yield a compound havingFormula IV wherein R₂ is hydrogen. During cleavage, the linkages betweenmonomeric subunits are converted from phosphotriester,thiophosphotriester, or dithiophosphotriester linkages tophosphodiester, phosphorothioate, or phosphorodithioate linkages.

[0214] Without intending that the invention be bound by any particulartheory, it is believed that the loss of the oxygen or sulfur protectinggroup where X₄ is an alkanoyl (e.g., acetyl) group occurs via afragmentation mechanism, illustrated in Scheme I below for embodimentswherein moiety A is phenyl with the group —OX₄ (exemplified as an acetylgroup) in the para position:

[0215] In this mechanism, a nucleophile (for example, ammonia) firstattacks the carbonyl carbon of the acetoyl group. The resonant movementof electrons as depicted in Scheme I above is believed to cause the lossof the oxygen or sulfur protecting group via a fragmentation, therebyforming a phosphodiester, phosphorothioate, or phosphorodithioatelinkage. The other products of the deprotection are NU—C(═O)—CH₃,p-quinone, and ethylene gas.

[0216] The mechanism for embodiments wherein moiety A is phenyl with thegroup —OX₄ (exemplified as an acetyl group) attached to the orthoposition is shown below in Scheme II:

[0217] The products of the deprotection are the unprotected linkage,Nu-C(═O)—CH₃, o-quinone, and ethylene gas.

[0218] In some preferred embodiments of the compounds of the invention,substituent X₄ is selected such that it facilitates attack by anucleophile, or a base. Accordingly, X₄ can be any of a variety ofsubstituents, provided that it does not otherwise interfere with themethods of the invention. Preferred non-silyl X₄ groups include alkarylgroups, sulfoxyl groups, sulfonyl groups, thio groups, substitutedsulfoxyl groups, substituted sulfonyl groups, or substituted thiogroups, wherein the substituents are selected from the group consistingof alkyl, aryl, or alkaryl. More preferred non-silyl X₄ groups includecompounds of formula —C(═O)—(O)_(aa)—R₄₀ where aa is 0 or 1 and R₄₀ islower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl, aryl,aralkyl or heteroaryl groups are optionally substituted with one or morealkyl, aryl, aralkyl, halo or acetyl groups. Particularly preferred X₄groups include acetyl (—C(═O)—CH₃), benzoyl (—C(═O)—Ph), phenylacetyl(—C(═O)—CH₂—Ph) and levulinyl (—C(═O)—(CH₂) ₂—C(═O) —CH₃) groups.

[0219] In one embodiment of the invention the moiety —OX₄ forms acarbonate or substituted carbonate group. In some preferred embodiments,X₄ has the formula —C(═O)—(O)_(aa)—R₄₀ where aa is 1 and R₄₀ is loweralkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl, aryl, aralkylor heteroaryl groups are optionally substituted with one or more alkyl,aryl, aralkyl, halo or acetyl groups. Carbonate protecting groups arediscussed in for example, Green and Wuts, Protective Groups in OrganicSynthesis, 2d edition, John Wiley & Sons, New York, 1991, pages 104-105et al., incorporated herein by reference.

[0220] X₄ can also be a group of formula a group of formula—(—CH₂—CH₂—)_(d)Si(R₉)₃ where d is 0 or 1, and each R₉ is,independently, alkyl having 1 to about 10 carbon atoms, or aryl having 6to about 10 carbon atoms. While not wishing to be bound by a particulartheory, it is believed that the loss of the oxygen or sulfur where X₄ isa trisubstituted silyl moiety, occurs via a fragmentation mechanism,illustrated in Scheme III below for embodiments wherein A is phenyl with—OX₄ at the para position:

[0221] In this mechanism, a nucleophile attacks the silyl silicom atom,and the resonant movement of electrons as depicted in Scheme III aboveis believed to cause the loss of the oxygen or sulfur protecting groupvia a fragmentation mechanism, thereby forming a phosphodiester,phosphorothioate, or phosphorodithioate linkage. The other products ofthe deprotection are believed to be ethylene gas, p-quinone, and acompound of formula Nu—Si(R₉)₃. For embodiments wherein the moiety(R₉)₃Si—is in the ortho position of the phenyl ring, the analogousfragmentation is beleived to result in the same products, except for theproduction of o-quinone instead of p-quinone. For embodiments wherein dis 1, it is believed that a similar fragmentation mechanism wouldproduce the same products, and one additional mole of ethylene.

[0222] A wide variety of bases or nucleophiles can be used to initiatethe fragmentation of the oxygen or sulfur protecting groups describedherein. These include ammonium hydroxide, fluoride ion, alkyl amines,aqueous bases, and alkyl amines in combination with ammonium hydroxide.The resulting products include phosphate, phosphorothioate, andphosphorodithioate containing compounds.

[0223] Contact with fluoride ion preferably is effected in a solventsuch as tetrahydrofuran, acetonitrile, dimethoxyethane, or water.Fluoride ion preferably is provided in the form of one or more saltsselected from tetraalkylammonium fluorides (e.g., tetrabutylammoniumfluoride (TBAF)), potassium fluoride, or cesium fluoride.

[0224] Preferably, conditions for removal of the oxygen or sulfurprotecting group via fragmentation mechanisms described above alsoeffect cleavage of the oligomeric compound from the solid support.

[0225] The methods of the present invention are useful for thepreparation of oligomeric compounds from monomeric or oligomeric amiditesynthons, for example synthons having Formula II. The internucleosidelinkages of such oligomeric amidite synthons, represented by moiety M inthe compounds and methods described herein, can be any internucleosidelinkage as is known in the art, including phosphorus based linkinggroups such as phosphite, phosphodiester, phosphorothioate, andphosphorodithioate linkages, and other linkages known in the art. Suchlinkages can be protected, i.e., they can bear, for example, phosphateprotecting groups. Included with the definition of internucleosidelinkages are groups described herein, having the Formula:

[0226] In preferred embodiments, the methods of the invention are usedfor the preparation of oligomeric compounds including oligonucleotidesand their analogs. As used herein, the term “oligonuclotide analog”means compounds that can contain both naturally occurring (i.e.“natural”) and non-naturally occurring (“synthetic”) moieties, forexample, nucleosidic subunits containing modified sugar and/ornucleobase portions. Such oligonucleotide analogs are typicallystructurally distinguishable from, yet functionally interchangeablewith, naturally occurring or synthetic wild type oligonucleotides. Thus,oligonucleotide analogs include all such structures which functioneffectively to mimic the structure and/or function of a desired RNA orDNA strand, for example, by hybridizing to a target. The term syntheticnucleoside, for the purpose of the present invention, refers to amodified nucleoside. Representative modifications include modificationof a heterocyclic base portion of a nucleoside to give a non-naturallyoccurring nucleobase, a sugar portion of a nucleoside, or bothsimultaneously.

[0227] Representative nucleobases useful in the compounds and methodsdescribed herein include adenine, guanine, cytosine, uridine, andthymine, as well as other non-naturally occurring and naturalnucleobases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl andother alkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 5-halo uracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil,8-halo, oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substitutedadenines and guanines, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine. Further naturally and non naturallyoccurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808(Merigan, et al.), in chapter 15 by Sanghvi, in Antisense Research andApplication, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, inEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613-722 (see especially pages 622 and 623, and in the ConciseEncyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed.,John Wiley & Sons, 1990, pages 858-859, Cook, P. D., Anti-Cancer DrugDesign, 1991, 6, 585-607, each of which are hereby incorporated byreference in their entirety. The term ‘nucleosidic base’ is furtherintended to include heterocyclic compounds that can serve as likenucleosidic bases including certain ‘universal bases’ that are notnucleosidic bases in the most classical sense but serve as nucleosidicbases. Especially mentioned as a universal base is 3-nitropyrrole.

[0228] As used herein the term “2′-substituent group” denotes groupsattached to the 2′ position of the ribosyl moiety, with or without anoxygen atom.

[0229] Preferred 2′-substituent groups described herein are representedin the compounds described herein by the variable R₁, which can beindependently, H, hydroxyl, C₁-C₂₀ alkyl, C₃-C₂₀ alkenyl, C₂-C₂₀alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso, nitrile,trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, or polyether;

[0230] or R₁ is a group of formula Z—R₂₂—(R₂₃)_(v);

[0231] Z is O, S, NH, or N—R₂₂—(R₂₃)_(v);

[0232] R₂₂ is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl;

[0233] R₂₃ is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl,nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl,S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl,S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido,hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide,disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reportermolecule, conjugate, polyamine, polyamide, polyalkylene glycol,polyether, a group that enhances the pharmacodynamic properties ofoligonucleotides, or a group that enhances the pharmacokineticproperties of oligonucleotides;

[0234] v is from 0 to about 10;

[0235] or R₁ has the formula:

[0236] y1 is 0 or 1;

[0237] y2 is independently 0 to 10;

[0238] y3 is 1 to 10;

[0239] E is C₁-C₁₀ alkyl, N(Q₁) (Q₂) or N═C(Q₁) (Q₂);

[0240] each Q₁ and Q₂ is, independently, H, C₁-C₁₀ alkyl, substitutedalkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered oruntethered conjugate group, a linker to a solid support; or Q₁ and Q₂,together, are joined in a nitrogen protecting group or a ring structurethat can include at least one additional heteroatom selected from N andO;

[0241] or R₁ has one of formula XI or XII:

[0242]  wherein

[0243] Z₀ is O, S, or NH;

[0244] q₁ is from 0 to 10;

[0245] q² is from 1 to 10;

[0246] q³ is 0 or 1;

[0247] q⁴ is, 0, 1 or 2;

[0248] Z₄ is OM₁, SM₁, or N(M₁)₂;

[0249] each M₁ is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C(═NH)N(H)M₂, C(═O)N(H)M₂ or OC(═O)N(H)M₂;

[0250] M₂ is H or C₁-C₈ alkyl;

[0251] Z₁, Z₂ and Z₃ comprise a ring system having from about 4 to about7 carbon atoms, or having from about 3 to about 6 carbon atoms and 1 or2 hetero atoms wherein said hetero atoms are selected from oxygen,nitrogen and sulfur, and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic; and

[0252] Z₅ is alkyl or haloalkyl having 1 to about 10 carbon atoms,alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q₁) (Q₂), OQ₁,halo, SQ₁ or CN.

[0253] Representative 2′-O- sugar substituents of formula XI aredisclosed in U.S. patent application Ser. No.: 09/130,973, filed Aug. 7,1998, entitled Capped 2′-Oxyethoxy Oligonucleotides, hereby incorporatedby reference in its entirety.

[0254] Representative cyclic 2′-O- sugar substituents of formula XII aredisclosed in U.S. patent application Ser. No.: 09/123,108, filed Jul.27, 1998, entitled RNA Targeted 2′-Modified Oligonucleotides that areConformationally Preorganized, hereby incorporated by reference in itsentirety.

[0255] One particularly preferred group includes 2′-methoxyethoxy[2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE] (Martinet al., Helv. Chim. Acta, 1995, 78, 486), i.e., an alkoxyalkoxy group. Afurther referred modification includes 2′-dimethylaminooxyethoxy, i.e.,a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inco-owned U.S. patent application Ser. No. 09/016,520, filed on Jan. 30,1998, the contents of which are herein incorporated by reference. Otherpreferred modifications include 2′-methoxy (2′-O—CH₃) and2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂).

[0256] Further preferred 2′-sugar modifications amenable to the presentinvention include fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy,protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, andpolyethers of the formula (O-alkyl)_(m) where m is 1 to about 10.Preferred among these polyethers are linear and cyclic polyethyleneglycols (PEGs), and (PEG)-containing groups, such as crown ethers andthose which are disclosed by Ouchi, et al., Drug Design and Discovery1992, 9, 93, Ravasio, et al., J. Org. Chem. 1991, 56, 4329, and Delgardoet. al., Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9,249, each of which are hereby incorporated by reference in theirentirety. Further sugar modifications are disclosed in Cook, P. D.,Anti-Cancer Drug Design, 1991, 6, 585-607. Fluoro, O-alkyl,O-alkylamino, O-alkyl imidazole, O-alkylaminoalkyl, and alkyl aminosubstitution is described in U.S. patent application Ser. No.08/398,901, filed Mar. 6, 1995, entitled Oligomeric Compounds havingPyrimidine Nucleotide(s) with 2′ and 5′ Substitutions, herebyincorporated by reference in its entirety.

[0257] Additional 2′ sugar modifications amenable to the presentinvention include 2′-SR and 2′-NR₂ groups, where each R is,independently, hydrogen, a protecting group or substituted orunsubstituted alkyl, alkenyl, or alkynyl. 2′-SR nucleosides aredisclosed in U.S. Pat. No. 5,670,633, issued Sep. 23, 1997, herebyincorporated by reference in its entirety. The incorporation of 2′-SRmonomer synthons are disclosed by Hamm et al., J. Org. Chem., 1997, 62,3415-3420. 2′-NR₂ nucleosides are disclosed by Goettingen, M., J. Org.Chem., 1996, 61, 6273-6281; and Polushin et al., Tetrahedron Lett.,1996, 37, 3227-3230.

[0258] Sugars having O-substitutions on the ribosyl ring are alsoamenable to the present invention. Representative substitutions for ringO include S, CH₂, CHF, and CF₂, see, e.g., Secrist, et al., Abstract 21,Program & Abstracts, Tenth International Roundtable, Nucleosides,Nucleotides and their Biological Applications, Park City, Utah,September 16-20, 1992, hereby incorporated by reference in its entirety.Additional modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide. Forexample, one additional modification of the oligonucleotides of theinvention involves chemically linking to the oligonucleotide one or moremoieties or conjugates which enhance the activity, cellular distributionor cellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharanet al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), athiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259,327; Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

[0259] As used herein, the term “alkyl” includes but is not limited tostraight chain, branch chain, and alicyclic hydrocarbon groups. Alkylgroups of the present invention may be substituted. Representative alkylsubstituents are disclosed in U.S. Pat. No. 5,212,295, at column 12,lines 41-50, hereby incorporated by reference in its entirety. As usedherein, the term “lower alkyl” is intended to mean alkyl having 6 orfewer carbons.

[0260] As used herein, the term “ara_kyl” denotes alkyl groups whichbear aryl groups, for example, benzyl groups. The term “alkaryl” denotesaryl groups which bear alkyl groups, for example, methylphenyl groups.As used herein the term “aryl” denotes aromatic cyclic groups includingbut not limited to phenyl, naphthyl, anthracyl, phenanthryl, andpyrenyl.

[0261] As used herein, the term “alkanoyl” has its accustomed meaning asa group of formula —C(═O)-alkyl. A preferred alkanoyl group is theacetyl group.

[0262] In general, the term “hetero” denotes an atom other than carbon,preferably but not exclusively N, O, or S. Accordingly, the term“heterocycloalkyl” denotes an alkyl ring system having one or moreheteroatoms (i.e., non-carbon atoms). Preferred heterocycloalkyl groupsinclude, for example, morpholino groups. As used herein, the term“heterocycloalkenyl” denotes a ring system having one or more doublebonds, and one or more heteroatoms. Preferred heterocycloalkenyl groupsinclude, for example, pyrrolidino groups.

[0263] In some embodiments of the invention, A is a monocyclic orbicyclic aromatic ring system. Suitable monocyclic or bicyclic aromaticring systems include phenyl, naphthyl, pyridyl, furyl and indolyl.

[0264] In some preferred embodiments of the compounds and methods of theinvention, R₁₁ and R₁₂ can be, together with the carbon atoms to whichthey are attached, an optionally substituted aliphatic or aromatic ringhaving from 4 to 6 ring atoms. Examples of such rings include phenyl andnaphthyl. Examples of substituents for such rings include halogen,hydroxyl, alkyl, and acetyl groups. In more preferred embodiments, R₁₁and R₁₂ are each H.

[0265] In some preferred embodiments of the invention R₂, or R₃ can be alinker connected to a solid support. Solid supports are substrates whichare capable of serving as the solid phase in solid phase syntheticmethodologies, such as those described in Caruthers U.S. Pat. Nos.4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418;

[0266] and Koster U.S. Pat. Nos. 4,725,677 and Re. 34,069.

[0267] Linkers are known in the art as short molecules which serve toconnect a solid support to functional groups (e.g., hydroxyl groups) ofinitial synthon molecules in solid phase synthetic techniques. Suitablelinkers are disclosed in, for example, Oligonucleotides And Analogues APractical Approach, Ekstein, F. Ed., IRL Press, N.Y., 1991, Chapter 1,pages 1-23.

[0268] Solid supports according to the invention include those generallyknown in the art to be suitable for use in solid phase methodologies,including, for example, controlled pore glass (CPG), oxalyl-controlledpore glass (see, e.g., Alul, et al., Nucleic Acids Research 1991, 19,1527, hereby incorporated by reference in its entirety), TentaGelSupport—an aminopolyethyleneglycol derivatized support (see, e.g.,Wright, et al., Tetrahedron Letters 1993, 34, 3373, hereby incorporatedby reference in its entirety) and Poros—a copolymer ofpolystyrene/divinylbenzene.

[0269] In some preferred embodiments of the invention R₂, R₃ or R_(3a)can be a hydroxyl protecting group. A wide variety of hydroxylprotecting groups can be employed in the methods of the invention.Preferably, the protecting group is stable under basic conditions butcan be removed under acidic conditions. In general, protecting groupsrender chemical functionalities inert to specific reaction conditions,and can be appended to and removed from such functionalities in amolecule without substantially damaging the remainder of the molecule.Representative hydroxyl protecting groups are disclosed by Beaucage, etal., Tetrahedron 1992, 48, 2223-2311, and also in Greene and Wuts,Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley &Sons, New York, 1991, each of which are hereby incorporated by referencein their entirety.

[0270] Preferred protecting groups used for R₂, R₃ and R_(3a) includedimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl(Pixyl)and 9-(p-methoxyphenyl)xanthen-9-yl(Mox). The R₂ or R₃ group can beremoved from oligomeric compounds of the invention by techniques wellknown in the art to form the free hydroxyl. For example, dimethoxytritylprotecting groups can be removed by protic acids such as formic acid,dichloroacetic acid, trichloroacetic acid, p-toluene sulphonic acid orwith Lewis acids such as for example zinc bromide. See for example,Greene and Wuts, supra.

[0271] In some preferred embodiments of the invention amino groups areappended to alkyl or to other groups such as, for example, to 2′-alkoxygroups. Such amino groups are also commonly present in naturallyoccurring and non-naturally occurring nucleobases. It is generallypreferred that these amino groups be in protected form during thesynthesis of oligomeric compounds of the invention. Representative aminoprotecting groups suitable for these purposes are discussed in Greeneand Wuts, Protective Groups in Organic Synthesis, Chapter 7, 2d ed, JohnWiley & Sons, New York, 1991. Generally, as used herein, the term“protected” when used in connection with a molecular moiety such as“nucleobase” indicates that the molecular moiety contains one or morefunctionalities protected by protecting groups.

[0272] Sulfurizing agents used during oxidation to form phosphorothioateand phosphorodithioate linkages include Beaucage reagent (see e.g. Iyer,R. P., et.al., J. Chem. Soc., 1990, 112, 1253-1254, and Iyer, R. P.,et.al., J. Org. Chem., 1990, 55, 4693-4699); tetraethylthiuram disulfide(see e.g., Vu, H., Hirschbein, B. L., Tetrahedron Lett., 1991, 32,3005-3008); dibenzoyl tetrasulfide (see e.g., Rao, M. V., et.al.,Tetrahedron Lett., 1992, 33, 4839-4842); di(phenylacetyl)disulfide (seee.g., Kamer, P. C. J., Tetrahedron Lett., 1989, 30, 6757-6760);Bis(O,O-diisopropoxy phosphinothioyl)disulfids (see Stec et al.,Tetrahedron Lett., 1993, 34, 5317-5320);3-ethoxy-1,2,4-dithiazoline-5-one (see Nucleic Acids Research, 1996 24,1602-1607, and Nucleic Acids Research, 1996 24, 3643-3644);Bis(p-chlorobenzenesulfonyl)disulfide (see Nucleic Acids Research, 199523, 4029-4033); sulfur, sulfur in combination with ligands like triaryl,trialkyl, triaralkyl, or trialkaryl phosphines. The foregoing referencesare hereby incorporated by reference in their entirety.

[0273] Useful oxidizing agents used to form the phosphodiester orphosphorothioate linkages include iodine/tetrahydrofuran/ water/pyridineor hydrogen peroxide/water or tert-butyl hydroperoxide or any peracidlike m-chloroperbenzoic acid. In the case of sulfurization thereaction-is performed under anhydrous conditions with the exclusion ofair, in particular oxygen whereas in the case of oxidation the reactioncan be performed under aqueous conditions.

[0274] Oligonucleotides or oligonucleotide analogs according to thepresent invention hybridizable to a specific target preferably comprisefrom about 5 to about 50 monomer subunits. It is more preferred thatsuch compounds comprise from about 10 to about 30 monomer subunits, with15 to 25 monomer subunits being particularly preferrred. When used as“building blocks” in assembling larger oligomeric compounds (i.e., assynthons of Formula II), smaller oligomeric compounds are preferred.Libraries of dimeric, trimeric, or higher order compounds of generalFormula II can be prepared for use as synthons in the methods of theinvention. The use of small sequences synthesized via solution phasechemistries in automated synthesis of larger oligonucleotides enhancesthe coupling efficiency and the purity of the final oligonucloetides.See for example: Miura, K., et al., Chem. Pharm. Bull., 1987, 35,833-836; Kumar, G., and Poonian, M. S., J. Org. Chem., 1984, 49,4905-4912; Bannwarth, W., Helvetica Chimica Acta, 1985, 68, 1907- 1913;Wolter, A., et al., nucleosides and nucleotides, 1986, 5, 65-77, each ofwhich are hereby incorporated by reference in their entirety.

[0275] In one aspect of the invention, the compounds of the inventionare used to modulate RNA or DNA, which code for a protein whoseformation or activity it is desired to modulate. The targeting portionof the composition to be employed is, thus, selected to be complementaryto the preselected portion of DNA or RNA, that is to be hybridizable tothat portion.

[0276] In some preferred embodiments of the methods of the invention,compounds of Formula II are prepared by reaction of a protectednucleoside having Formula V:

[0277] and a phosphine compound of Formula VI:

[0278] in the presence of an acid. Suitable acids include those known inthe art to be useful for coupling of phosphoramidites, including, forexample, tetrazole, substituted tetrazoles, dicyanoimidazole, ordiisopropylammonium tetrazolide.

[0279] In some preferreed embodiments, compounds of Formula VI areprepared by reacting an alcohol having the Formula XX:

[0280] with phosphorus trichloride, and reacting the resultant product,Cl₂P—X₁—(CH₂)₂—X₅—C₆H₄—OX₄, with at least two equivalents of an aminehaving the formula [(R₆)₂N]₂NH. Each of the R₆ groups can be the same ordifferent, and are preferably alkyl having 1 to about 10 carbon atoms,more preferably 1 to 6 carbon atoms, with 3 carbon atoms, andparticularly isopropyl groups, being especially preferred.

[0281] In further preferred embodiments, compounds of Formula II can beprepared by reaction of a protected nucleoside of Formula V with achlorophosphine compund of formula ClP(R₅)₂, where R₅ is preferablyisopropylamino, followed by reaction with a compound of Formula XX inthe presence of an acid, for example 1-H tetrazole, substitutedtetrazoles, or dicyanoimidazole, with 1-H tetrazole being preferred.

[0282] In some particularly preferred embodiments of the foregoingmethods, X₄ is benzoyl, acetyl or levulinyl, A is phenyl with the moiety—OX₄ being in the ortho or para position thereof, with the orthoposition being more preferred, or A is naphthalene connected to X₅ atthe 1-position, with the moiety —OX₄ being in the 5- or 6-position ofthe naphthalene ring.

[0283] In the compounds and methods of the present inventon, X₁ and X₂can each independently be O or S. Thus, compounds having chiralphosphorus linkages are contemplated by the present invention. See Stec,W. J., and Lesnikowski, Z. J., in Methods in Molecular Biology Vol. 20:Protocols for Oligonucleotides and Analogs, S. Agrawal, Ed., HumanaPress, Totowa, N.J. (1993), at Chapter 14. See also Stec, W. J. et al.,Nucleic Acids Research, Vol. 19, No. 21, 5883-5888 (1991); and EuropeanPatent Application EP 0 506 242 A1, each of which are herebyincorporated by reference in their entirety.

[0284] Also provided in preferred embodiments of the invention arecompounds having the general Formula VII:

[0285] wherein X₁, A, X₄ and X₅ and D are as defined above.

[0286] In particularly preferred embodiments, the compounds of theinvention have the Formula II:

[0287] wherein:

[0288] X₄, M, X₁, R₁, X₂, R₃, B, n, and R₅ are defined as above. In someespecially preferred embodiments of the compounds of the inventionhaving formula II above, X₄ is benzoyl, acetyl or levulinyl, or a groupof formula —(CH₂—CH₂)_(d)Si (R₉)₃ where d is 0 or 1; A is phenyl havingthe moiety —OX₄ is in the ortho or para position, with the orthoposition being preferred, R₅ is diisopropylamino, and n is 0.

[0289] The oligomeric compounds of the invention can be used indiagnostics, therapeutics and as research reagents and kits. They can beused in pharmaceutical compositions by including a suitablepharmaceutically acceptable diluent or carrier. They further can be usedfor treating organisms having a disease characterized by the undesiredproduction of a protein. The organism should be contacted with anoligonucleotide having a sequence that is capable of specificallyhybridizing with a strand of nucleic acid coding for the undesirableprotein. Treatments of this type can be practiced on a variety oforganisms ranging from unicellular prokaryotic and eukaryotic organismsto multicellular eukaryotic organisms. Any organism that utilizesDNA-RNA transcription or RNA-protein translation as a fundamental partof its hereditary, metabolic or cellular control is susceptible totherapeutic and/or prophylactic treatment in accordance with theinvention. Seemingly diverse organisms such as bacteria, yeast,protozoa, algae, all plants and all higher animal forms, includingwarm-blooded animals, can be treated. Further, each cell ofmulticellular eukaryotes can be treated, as they include both DNA-RNAtranscription and RNA-protein translation as integral parts of theircellular activity. Furthermore, many of the organelles (e.g.,mitochondria and chloroplasts) of eukaryotic cells also includetranscription and translation mechanisms. Thus, single cells, cellularpopulations or organelles can also be included within the definition oforganisms that can be treated with therapeutic or diagnosticoligonucleotides.

[0290] As will be recognized, the steps of the methods of the presentinvention need not be performed any particular number of times or in anyparticular sequence. Additional objects, advantages, and novel featuresof this invention will become apparent to those skilled in the art uponexamination of the following examples thereof, which are intended to beillustrative and not intended to be limiting.

EXAMPLE Example 1 2-Acetoxyphenoxyethyl Alcohol

[0291] 2-(2-Hydroxyethoxy)phenol (308 g; 2 mol) was taken up in a 5 LErlnmeyer flask fitted with mechanical stirrer. Anhydrous acetone (4 L,dried over K₂CO₃), and potassium carbonate powder (290 g; 2.1 mol) wereadded, and the mixture was stirred vigourously. Acetic anhydride (207mL; 2.2 mol) was added from an additional funnel slowly over a period of1 hour. Stiiring was continued for 3 hours. TLC (CH₂Cl₂/MeOH : 9:1, v/v)showed disapperence of starting material. The reaction mixture wasfiltered, solid washed thoroughly with acetone (1 L). The combinedfractions was concentrated and purified by chromatography eluting withhexane and ethyl acetate (0% to 35% EtOAc; v/v). The product wasobtained as a colorless viscous oil. Yield 258-264 gms (70-72%)

Example 2 General Method for the Synthesis of Phosphoramidites

[0292] A 500 mL two-necked flask equipped with a magnetic stirrer, a gasinlet for argon, and a septum was assembled under an argon atmosphere.All glassware were dried at 120° C. for 1 hour. The flask was chargedwith bis(diisopropylamino) chlorophosphine (84.6 mmol), Hünigs base(diisopropylethylamine) (105.8 mmol) and anhydrous dichloromethane (250mL). With stirring, DMT-protected deoxyribonucleoside (70.5 mmol) wasadded as a solid over a period of 10 minutes. After 30 minutes, all thevolatiles were removed under vacuum (oil pump) and the residue dissolvedin anhydrous acetonitrile (150 mL). A solution of the2-acetoxyphenoxyethyl alcohol (105.7 mmol) in acetonitrile (100 mL) wasadded followed by 1H-tetrazole (63 mmol). Stirring was continued for afurther 1 hour. The reaction mixture was then concentrated, and theresidue redissolved in dichloromethane (500 mL), washed with sodiumbicarbonate solution and dried (Na₂SO₄). Concentration of the driedsolution afforded the crude material which was purified by silica gelflash chromatography. The fractions corresponding to the amidites werecombined and concentrated to afford the pure product as a foammy solid.Yields 65-80%.

Example 3 Preparation of 2-acetoxyphenoxyethyl N,N-diisopropylphosphoramidite

[0293] A 500 mL three-necked flask equipped with a magnetic stirrer, aglass stopper and an inlet for argon was assembled under argonatmosphere. All glassware was dried in an oven at 120° C. for 1 hour.The reaction flask was charged with anhydrous ether (150 mL) andphosphorous trichloride (9.27 g; 67.5 mmol). 2-Acetoxyphenoxyethylalcohol (50 mmol) in ether (100 mL) was added to the reaction flaskslowly with stirring at 0° C. (ice cooling) using pressure-equalizedaddition funnel. After addition was complete, ice bath was removed andthe reaction was stirred for three hours. The reaction mixture then wastransferred to a 500 mL flask and concentrated under reduced pressure.To this product in anhydrous ether (200 mL) was added diisopropylamine(57.7 mL) at 0° C. under argon. After the addition was complete,stirring was continued at room temperature for 16 hours (overnight). Thereaction mixture was filtered and concentrated to afford the product.

Example 4 General Method for the Synthesis of Phosphoramidites

[0294] A 250 mL two-necked flask equipped with a magnetic stirrer, a gasinlet for argon, and a septum was assembled under an argon atmosphere.All glassware was dried at 120° C. for 1 hour. The flask was chargedwith 5′-O-DMT nucleoside (7 mmol) and 1H-tetrazole (5.6 mmol). Anhydrousacetonitrile (50 mL) was added. To this stirred mixture under argon atroom temperature was added a solution of 2-acetoxyphenoxyethylN,N-diispropylphosphoramidite (10.5 mmol) in acetonitrile (50 mL). Usualworkup followed by purification afforded the phosphoramidites.

Example 5 Synthesis of T-T Phosphorothioate Dimer

[0295] 100 milligram (4 mmole) of 5′-O-Dimethoxytritylthymidine bondedto CPG (controlled pore glass) through an ester linkage was taken in aglass reactor, and a dichloromethane solution of 2% dichloroacetic acid(volume/volume) was added to deprotect the 5′-hydroxyl group. Theproduct was washed with dichloromethane and then with acetonitrile.Then, a 0.2 M solution of5′-O-(4,40-dimethoxytrityl)thymidine-3′-O-(2-acetoxyphenoxyethyl)-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrilewere added, and reacted at room temperature for 5 minutes. The productwas washed with acetonitrile, and then a 0.05 M solution of Beaucagereagent in acetonitrile was added and reacted at room temperature for 5minutes. This sulfurization step was repeated one more time for 5minutes. The support was washed with acetonitrile and then a solution ofacetic anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF wasadded to cap the unreacted 5′-hydroxyl group. The product was washedwith acetonitrile.

[0296] The carrier containing the compound was treated with 30% aqueousammonium hydroxide solution for 90 minutes. The aqueous solution wasfiltered, concentrated under reduced pressure to give phosphorothioatedimer of T-T.

Example 6 Synthesis of C-T Phosphorothioate Dimer

[0297] 100 milligram (4 mmole) of 5′-O-Dimethoxytritylthymidine bondedto CPG (controlled pore glass) through an ester linkage was taken in aglass reactor, and a dichloromethane solution of 2% dichloroacetic acid(volume/volume) was added to deprotect the 5′-hydroxyl group. Theproduct was washed with acetonitrile. Then, a 0.2 M solution ofN⁴-Benzoyl-5′-O-(4,40-dimethoxytrityl)-2′-deoxycytidine-3′-O-(2-acetoxyphenoxyethyl)-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrilewere added, and reacted at room temperature for 5 minutes. The productwas washed with acetonitrile, and then a 0.05 M solution of Beaucagereagent in acetonitrile was added and reacted at room temperature for 5minutes. This sulfurization step was repeated one more time for 5minutes. The support was washed with acetonitrile and then a solution ofacetic anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF wasadded to cap the unreacted 5′-hydroxyl group. The product was washedwith acetonitrile.

[0298] The carrier containing the compound was treated with 30% aqueousammonium hydroxide solution for 90 minutes and then incubated at 55° C.for 12 hours. The aqueous solution was filtered, concentrated underreduced pressure and then treated at room temperature with 1.0 Msolution of tetra-n-butyl ammonium fluoride in THF to give aphosphorothioate dimer of dC-T.

Example 7 Synthesis of 5′-TTTTTTT-3′ Phosphorothioate Heptamer

[0299] 50 milligram (2 mmole) of 5′-O-Dimethoxytritylthymidine bonded toCPG (controlled pore glass) through an ester linkage is taken in a glassreactor, and a dichloromethane solution of 2% dichloroacetic acid(volume/volume) is added to deprotect the 5′-hydroxyl group. The productis washed with acetonitrile. Then, a 0.2 M solution of5′-O-(4,4′-dimethoxytrityl)thymidine-3′-O-(2-acetoxyphenoxyethyl)-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrile isadded, and reacted at room temperature for 5 minutes. The product iswashed with acetonitrile, and then a 0.05 M solution of Beaucage reagentin acetonitrile is added and reacted at room temperature for 5 minutes.This sulfurization step is repeated one more time for 5 minutes. Thesupport is washed with acetonitrile and then a solution of aceticanhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added tocap the unreacted 5′-hydroxyl group. The product is washed withacetonitrile.

[0300] This complete cycle is repeated five more times to get thecompletely protected thymidine heptamer. The carrier containing thecompound is treated with 30% aqueous ammonium hydroxide solution for 90minutes at room temperature. The aqueous solution is filtered,concentrated under reduced pressure to give a phosphorothioate heptamerof TTTTTTT.

Example 8 Synthesis of 5′-d(GACTT)-3′ Phosphorothioate Tetramer

[0301] 50 milligram (2 mmole) of 5′-O-Dimethoxytritylthymidine bonded toCPG (controlled pore glass) through an ester linkage was taken in aglass reactor, and a dichloromethane solution of 2% dichloroacetic acid(volume/volume) was added to deprotect the 5′-hydroxyl group. Theproduct was washed with acetonitrile. Then, a 0.2 M solution of5′-O-(4,4′-dimethoxytrityl)thymidine-3′-O-(2-acetoxyphenoxyethyl)-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrile wasadded, and reacted at room temperature for 5 minutes. The product waswashed with acetonitrile, and then a 0.05 M solution of Beaucage reagentin acetonitrile was added and reacted at room temperature for 5 minutes.This sulfurization step was repeated one more time for 5 minutes. Thesupport was washed with acetonitrile and then a solution of aceticanhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF was added tocap the unreacted 5′-hydroxyl group. The product was washed withacetonitrile.

[0302] A dichloromethane solution of 2% dichloroacetic acid(volume/volume) was added to deprotect the 5′-hydroxyl group. Theproduct was washed with acetonitrile. Then, a 0.2 M solution ofN⁴-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine-3′-O-(2-acetoxyphenoxyethyl)-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrilewere added, and reacted at room temperature for 5 minutes. The productwas washed with acetonitrile, and then a 0.05 M solution of Beaucagereagent in acetonitrile was added and reacted at room temperature for 5minutes. This sulfurization step was repeated one more time for 5minutes. The support was washed with acetonitrile and then a solution ofacetic anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF wasadded to cap the unreacted 5′-hydroxyl group. The product was washedwith acetonitrile.

[0303] A dichloromethane solution of 2% dichloroacetic acid(volume/volume) was added to deprotect the 5′-hydroxyl group. Theproduct was washed with acetonitrile. Then, a 0.2 M solution ofN⁶-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine-3′-O-(2-acetoxyphenoxyethyl)-N,N-diisopropylphosphoramidite)in anhydrous acetonitrile and a 0.4 M solution of 1H-tetrazole inacetonitrile was added, and reacted at room temperature for 5 minutes.The product was washed with acetonitrile, and then a 0.05 M solution ofBeaucage reagent in acetonitrile was added and reacted at roomtemperature for 5 minutes. This sulfurization step was repeated one moretime for 5 minutes. The support was washed with acetonitrile and then asolution of acetic anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF was added to cap the unreacted 5′-hydroxyl group. Theproduct-was washed with acetonitrile.

[0304] A dichloromethane solution of 2% dichloroacetic acid(volume/volume) was added to deprotect the 5′-hydroxyl group. Theproduct was washed with acetonitrile. Then, a 0.2 M solution ofN²-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine-3′-O-(2-acetoxyphenoxyethyl)-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4 M solution of 1H-tetrazole in acetonitrilewere added, and reacted at room temperature for 5 minutes. The productwas washed with acetonitrile, and then a 0.05 M solution of Beaucagereagent in acetonitrile were added and reacted at room temperature for 5minutes. This sulfurization step was repeated one more time for 5minutes. The support was washed with acetonitrile and then a solution ofacetic anhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF wasadded to cap the unreacted 5′-hydroxyl group. The product was washedwith acetonitrile.

[0305] The carrier containing the compound was treated with 30% aqueousammonium hydroxide solution for 90 minutes at room temperature and thenincubated at 55° C. for 16 hours. The aqueous solution was filtered, andconcentrated under reduced pressure to give a phosphorothioate tetramerof 5′-d (GACTT) -3′.

Example 9 Synthesis of Fully-modified5′-d(TCC-CGC-CTG-TGA-CAT-GCA-TT)-3′ Phosphorothioate 20-mer

[0306] The synthesis of the above sequence was performed on a PharmaciaOligoPilot II Synthesizer on a 620 mmole scale using the2-acetoxyphenoxyethyl phosphoramidites and Pharmacia's primar support.Detritylation was performed using 3% dichloroacetic acid indichloromethane (volume/volume). Sulfurization was performed using a 0.2M solution of Beaucage reagent in acetonitrile: for 2 minutes. At theend of synthesis, the support was washed with acetonitrile, cleaved,deprotected and purified in the usual manner.

Example 10 Synthesis of Fully-modified5′-d(GCC-CAA-GCT-GGC-ATC-CGT-CA)-3′ Phosphorothioate 20-mer

[0307] The synthesis of the above sequence was performed on a PharmaciaOligoPilot II Synthesizer on a 620 mmole scale using the2-acetoxyphenoxyethyl phosphoramidites and Pharmacia's primar support.Detritylation was performed using 3% dichloroacetic acid indichloromethane (volume/volume). Sulfurization was performed using a 0.2M solution of Beaucage reagent in acetonitrile: for 2 minutes. At theend of synthesis, the support was washed with acetonitrile, cleaved,deprotected and purified in the usual manner.

Example 11 Synthesis of Fully-modified5′-d(GCG-TTT-GCT-CTT-CTT-CTT-GCG)-3′ Phosphorothioate 21-mer

[0308] The synthesis of the above sequence was performed on a PharmaciaOligoPilot II Synthesizer on a 620 mmole scale using the2-acetoxyphenoxyethyl phosphoramidites and Pharmacia's primar support.Detritylation was performed using 3% dichloroacetic acid indichloromethane (volume/volume). Sulfurization was performed using a 0.2M solution of Beaucage reagent in acetonitrile: for 2 minutes. At theend of synthesis, the support was washed with acetonitrile, cleaved,deprotected and purified in the usual manner.

Example 12 Synthesis of Fully-modified5′-d(GTT-CTC-GCT-GGT-GAG-TTT-CA)-3′ Phosphorothioate 20-mer

[0309] The synthesis of the above sequence was performed on a PharmaciaOligoPilot II Synthesizer on a 620 mmole scale using the2-acetoxyphenoxyethyl phosphoramidites and Pharmacia's primar support.Detritylation was performed using 3% dichloroacetic acid in toluene(volume/volume). Sulfurization was performed using a 0.2 M solution ofphenylacetyl disulfide in acetonitrile:3-picoline (1:1 v/v) for 2minutes. At the end of synthesis, the support was washed withacetonitrile, cleaved, deprotected and purified in the usual manner.

Example 13 Synthesis of Fully-modified5′-d(TCC-CGC-CTG-TGA-CAT-GCA-TT)-3′ Phosphorothioate 20-mer

[0310] The synthesis of the above sequence was performed on a PharmaciaOligoPilot II Synthesizer on a 620 mmole scale using the2-acetoxyphenoxyethyl phosphoramidites and Pharmacia's primar support.Detritylation was performed using 3% dichloroacetic acid indichloromethane (volume/volume). Sulfurization was performed using a 0.2M solution of phenylacetyl disulfide reagent in acetonitrile:picoline(1:1 v/v) for 2 minutes. At the end of synthesis, the support was washedwith acetonitrile, cleaved, deprotected and purified in the usualmanner.

Example 14 Synthesis of Fully-modified5′-d(GCC-CAA-GCT-GGC-ATC-CGT-CA)-3′ Phosphorothioate 20-mer

[0311] The synthesis of the above sequence was performed on a PharmaciaOligoPilot II Synthesizer on a 620 mmole scale using the2-acetoxyphenoxyethyl phosphoramidites and Pharmacia's primar support.Detritylation was performed using 3% dichloroacetic acid indichloromethane (volume/volume). Sulfurization was performed using a 0.2M solution of phenylacetyl disulfide reagent in acetonitrile:picoline(1:1 v/v) for 2 minutes. At the end of synthesis, the support was washedwith acetonitrile, cleaved, deprotected and purified in the usualmanner.

Example 15 Synthesis of fully-Modified5′-d(GCG-TTT-GCT-CTT-CTT-CTT-GCG)-3′ Phosphorothioate 21-mer

[0312] The synthesis of the above sequence was performed on a PharmaciaOligoPilot II Synthesizer on a 620 mmole scale using the2-acetoxyphenoxyethyl phosphoramidites and Pharmacia's primar support.Detritylation was performed sing 3% dichloroacetic acid indichloromethane (volume/volume). Sulfurization was performed using a 0.2M solution of phenylacetyl disulfide reagent in acetonitrile:picoline(1:1 v/v) for 2 minutes. At the end of synthesis, the support was washedwith acetonitrile, cleaved, deprotected and purified in the usualmanner.

Example 16 Acetylation of 2-(2-hydroxyethoxy)phenol

[0313] A 4 liter flask equipped with a mechanical stirrer, and anadditional funnel is assembled under an atmosphere of argon. All theglassware is dried at 120° C. for 1 h. 2-(2-Hydroxyethoxyphenol) (508.8g, 3.3 mole) was added to the flask as a solid and dissolved inanhydrous methylene chloride (2.2 L). Triethylamine (1.38 L, 9.9 mole)was added slowly followed by the addition of acetic anhydride (934 mL;9.9 mole) at room temperature slowly over a period of 2 h. The reactionmixture becomes slightly warm. The reaction mixture was stirred at roomtemperature overnight. The reaction mixture was diluted with methylenechloride (1 L), washed with a solution of saturated sodium bicarbonate(till effervescence is complete), brine, dried over (MgSO₄) andconcentrated. The crude material was distilled to afford 716 g (91%,b.p. 133-135° C./0.1 mm) of the title compound as a colorless viscousliquid.

Example 17 Chemoselective Hydrolysis using Bisacetate of2-(2-hydroxyethoxy)phenol

[0314] A 1 liter flask equipped with a magnetic stirrer, a gas inlet forargon, and a septum was assembled under an atmosphere of argon. All theglassware was dried at 120° C. for 1 h. The bisacetate of2-(2-hydroxyethoxyphenol) (35 g) was added to the flask and dissolved inanhydrous tetra-hydrofuran (350 mL). n-Butanol (52.5 mL) was added tothe mixture followed by the addition of PLY Lipase (type II, Sigma). Thereaction mixture was stirred at room temperature for 48 h (HPLCmonitoring). The mixture was then filtered, the solid was washed withethyl acetate (400 ml) and the combined fractions concentrated.Purification of the material by flash chromatography gave the 28 g ofthe title compound as a colorless viscous liquid.

Example 18 Hydrolysis of the Bisacetate of 2-(2-hydroxyethoxy)phenolusing recycled enzyme

[0315] A 1 liter flask equipped with a magnetic stirrer, a gas inlet forargon, and a septum was assembled under an atmosphere of argon. All theglassware was dried at 120° C. for 1 h. The bisacetate of2-(2-hydroxyethoxyphenol) (35 g) was added to the flask and dissolved inanhydrous tetrahydrofuran (350 mL). n-Butanol (52.5 mL) was added to themixture followed by the addition of PLY Lipase (type II, Sigma). Thereaction mixture was stirred at room temperature for 48 h (HPLCmonitoring). The mixture was then filtered, the solid washed with ethylacetate (600 mL) and the combined fractions concentrated. Purificationof the material by flash column chromatography gave 26.8 g of the titlecompound as a colorless viscous liquid.

Example 19 Synthesis of 2Õ-(2-hydroxyethoxy)acetophenone

[0316] A 1 liter flask equipped with a magnetic stirrer, a gas inlet forargon, and a septum is assembled under an atmosphere of argon. All theglassware is dried at 120° C. for 1 h. 2Õ-Hydroxyacetophenone (13.6 g)is added to the flask and dissolved in xylene (350 mL). Ethylenecarbonate (17.6 g) is added to the mixture followed by the addtion ofsolid powdered potassium carbonate (55.2 g). The reaction mixture isstirred and refluxed for 12-15 h, cooled, filtered, and concentrated.Purification of the material by flash chromatography affords the titlecompound.

Example 20 Baeyer-Villiger oxidation of 2Õ-(2-hydroxyethoxy)acetophenone

[0317] A 250 mL flask equipped with a magnetic stirrer, a gas inlet forargon, and a septum is assembled under an atmosphere of argon. All theglassware is dried at 120° C. for 1 h. 2Õ-(2-Hydroxyethoxy)acetophenone(1.79 g, 10 mmol) is added to the flask and dissolved in anhydrousmethylene chloride (25 mL). To this stirred solution at room temperatureis added m-chloroperbenzoic acid (50-60% or any technical grade) as asolid. The reaction mixture is cooled to 5° C. and trifluoroacetic acidis added dropwise over a period of 5 min. The reaction mixture isprotected from light and allowed to warm to room temperature. After 5 h,the reaction mixture is diluted with methylene chloride (25 ml),filtered, and the filtrate washed with a solution of saturated sodiumcarbonate, brine and dried. Concentration and purification using flashcolumn chromatography affords the title compound.

Example 21 Synthesis of 4Õ-(2-hydroxyethoxy)acetophenone

[0318] A 1 L flask equipped with a magnetic stirrer, a gas inlet forargon, and a septum is assembled under an atmosphere of argon. All theglassware is dried at 120° C. for 1 h. 4Õ-(2-Acetoxyethoxy)acetophenone(22.22 g) is added to the flask and dissolved in methanol (150 mL).Powdered potassium cyanide (13 g) is added to the solution and stirredat room temperature for 3 h. The reaction mixture is concentrated to asolid, taken up in minimum amount of methylene chloride and passedthrough a pad of silica gel. Concentration of the eluate affords thetitle compound.

Example 22 Baeyer-Villiger oxidation of 4Õ-(2-hydroxyethoxy)acetophenone

[0319] A 250 mL flask equipped with a magnetic stirrer, a gas inlet forargon, and a septum is assembled under an atmosphere of argon. All theglassware is dried at 120° C. for 1 h. 4Õ-(2-Hydroxyethoxy)acetophenone(1.79 g, 10 mmol) is added to the flask, dissolved in anhydrousmethylene chloride (25 ml). To this stirred solution at room temperatureis added m-chloroperbenzoic acid (50-60% or any technical grade) as asolid. The reaction mixture is cooled to 5° C. and trifluoroacetic acidis added dropwise over a period of 5 min. The reaction mixture isprotected from light and allowed to warm to room temperature. After 5 h,the reaction mixture is diluted with methylene chloride (25 mL),filtered, and the filtrate washed with saturated sodium carbonatesolution, brine and dried. Concentration and purification using flashchromatography affords the title compound.

Example 23 Synthesis of Fully Protected Diol

[0320] A 250 mL flask equipped with a magnetic stirrer, a gas inlet forargon, and a septum is assembled under an atmosphere of argon. All theglassware is dried at 120° C. for 1 h. 2-(2-Hydroxyethoxy)phenol (7.71g) is added to the flask and dissolved in anhydrous methylene chloride(100 mL). Ethyl vinyl ether (3.97 g) is added to the solution followedby the addition of catalytic amount of PPTS. The reaction mixture isstirred at room temperature for 3 h. Triethylamine is added followed bythe addition of acetic anhydride. The mixture is stirred at roomtemperature for 6 h, concentrated, taken up in ethyl acetate (150 mL),washed with a solution of sodium bicarbonate, brine, dried andconcentrated. The crude title compound is used as such in the subsequentreaction.

Example 24 Hydrolysis of ethoxyethyl ether

[0321] A 250 mL flask equipped with a magnetic stirrer, a gas inlet forargon, and a septum is assembled under an atmosphere of argon. All theglassware is dried at 120° C. for 1 h. The fully protected2-(2-hydroxyethoxy)phenol is taken up in methylene chloride andn-propanol is added to it followed by the addition of catalytic amountof pyridinium tosylate at 5° C. After stirring the mixture for 12 h,work up and purification by flash column chromatography affords thetitle compound.

Example 25 Synthesis of bis tert-butyldimethylsilyl ether of2-(2-hydroxyethoxy)phenol

[0322] A 250 mL flask equipped with a magnetic stirrer, a gas inlet forargon, and a septum is assembled under an atmosphere of argon. All theglassware is dried at 120° C. for 1 h. 2-(2-Hydroxyethoxy)phenol (7.71g; 0.05 mole) is added to the flask and dissolved in anhydrous methylenechloride (100 mL). Triethylamine (20.2 g, 0.2 mole) is added to thesolution followed by the addition of tert-butyldimethylsilyl chloride(18.09 g; 0.12 mole). A catalytic amount of DMAP is added to acceleratethe reaction. The reaction mixture is stirred at room temperature for 12h and then worked up and purified by flash column chromatography to givethe title compound.

Example 26 Selective hydrolysis of bis tert-butyldimethylsilyl ether of2-(2-hydroxyethoxy)phenol

[0323] Bis tert-butyldimethylsilyl ether of 2-(2-hydroxy-ethoxy)phenol(0.01 mole) is taken up in methanol. Then 1 wt % (10 mg/mL) of solidiodine is added and the reaction monitored by tlc. Upon consumption ofthe alcoholic silyl ether, solid sodium metabisulfite is added andstirred until iodine color disappears. The methanolic solution isdiluted with methylene chloride (120 mL), washed with saturated sodiumbicarbonate, brine and dried. Purification by flash columnchromatography affords the title compound.

[0324] It is intended that each of the patents, applications, printedpublications, and other published documents mentioned or referred to inthis specification be herein incorporated by reference in theirentirety.

[0325] Those skilled in the art will appreciate that numerous changesand modifications may be made to the preferred embodiments of theinvention and that such changes and modifications may be made withoutdeparting from the spirit of the invention. It is therefore intendedthat the appended claims cover all such equivalent variations as fallwithin the true spirit and scope of the invention.

What is claimed is:
 1. A method for the preparation of an oligomericcompound comprising a moiety having the Formula I:

wherein: A is a monocyclic or bicyclic aromatic ring system; R₁₁ and R₁₂are each independently H, alkyl, aryl, heteroalkyl, heteroaryl, alkaryl,or aralkyl; or R₁₁ and R₁₂ together with the carbon atoms to which theyare attached form an optionally substituted aliphatic or aromatic ringhaving from 4 to 6 ring atoms; X₄ is alkaryl, aralkyl, sulfoxyl,sulfonyl, thio, substituted sulfoxyl, substituted sulfonyl, orsubstituted thio, wherein said substituent is alkyl, aryl, or alkaryl;or X₄ is a group of formula —C(═O)—(O)_(aa)—R₄₀ where aa is 0 or 1 andR₄₀ is lower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl,aryl, aralkyl or heteroaryl groups are optionally substituted with oneor more alkyl, aryl, aralkyl, halo or acetyl groups; or X₄ is a group offormula —(—CH₂—CH₂—)_(d)Si(R₉)₃ where d is 0 or 1; each R₉ is,independently, alkyl having 1 to about 10 carbon atoms, or aryl having 6to about 10 carbon atoms; X₁ and X₅ are each independently O or S;comprising: (a) providing a compound having the Formula II:

 wherein: each R₁, is, independently, H, hydroxyl, C₁-C₂₀ alkyl, C₃-C₂₀alkenyl, C₂-C₂₀ alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso,nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, or polyether; or R₁ is agroup of formula Z—R₂₂—(R₂₃)_(v); Z is O, S, NH, or N—R₂₂—(R₂₃)_(v); R₂₂is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl; R₂₃ is hydrogen,amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso,nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, polyether, a group thatenhances the pharmacodynamic properties of oligonucleotides, or a groupthat enhances the pharmacokinetic properties of oligonucleotides; v isfrom 0 to about 10; or R₁ has the formula:

y1 is 0 or 1; y2 is independently 0 to 10; y3 is 1 to 10; E is C₁-C₁₀alkyl, N(Q₁) (Q₂) or N═C(Q₁) (Q₂); each Q₁ and Q₂ is, independently, H,C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, dialkylaminoalkyl, a nitrogenprotecting group, a tethered or untethered conjugate group, a linker toa solid support; or Q₁ and Q₂, together, are joined in a nitrogenprotecting group or a ring structure that can include at least oneadditional heteroatom selected from N and O; or R₁ has one of formula XIor XII:

 wherein Z₀ is O, S, or NH; q¹ is from 0 to 10; q² is from 1 to 10; q³is 0 or 1; q⁴ is, 0, 1 or 2; Z₄ is OM₁, SM₁, or N(M₁)₂; each M₁ is,independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C(═NH)N(H)M₂,C(═O)N(H)M₂ or OC(═O)N(H)M₂; M₂ is H or C₁-C₈ alkyl; Z₁, Z₂ and Z₃comprise a ring system having from about 4 to about 7 carbon atoms, orhaving from about 3 to about 6 carbon atoms and 1 or 2 hetero atomswherein said hetero atoms are selected from oxygen, nitrogen and sulfur,and wherein said ring system is aliphatic, unsaturated aliphatic,aromatic, or saturated or unsaturated heterocyclic; and Z₅ is alkyl orhaloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having6 to about 14 carbon atoms, N(Q₁)(Q₂), OQ₁, halo, SQ₁ or CN; R₃ ishydrogen, a hydroxyl protecting group, or a linker connected to a solidsupport; each B, independently, is a naturally occurring ornon-naturally occurring nucleobase or a protected naturally occurring ornon-naturally occurring nucleobase; n is 0 to about 50; M is anoptionally protected internucleoside linkage; R₅ is —N(R₆)₂, or aheterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur,and oxygen; and R₆ is straight or branched chain alkyl having from 1 to10 carbons; and (b) reacting the compound of Formula II with a compoundhaving Formula III:

 wherein m is 0 to about 50; R_(3a) is hydrogen; R₂ is hydrogen, ahydroxyl protecting group, or a linker connected to a solid support,provided that R₂ and R_(3a) are not both simultaneously a linkerconnected to a solid support; to form the oligomeric compound.
 2. Themethod of claim 1 wherein A is phenyl or naphthalene.
 3. The method ofclaim 1 further comprising the step of oxidizing or sulfurizing theoligomeric compound.
 4. The method of claim 3 further comprisingdeprotecting the sulfurized or oxidized compound to form a furthercompound of Formula III.
 5. The method of claim 3 further comprising acapping step.
 6. The method of claim 4 further comprising the step ofcleaving the oligomeric compound from the solid support to produce acompound having the Formula IV:


7. The method of claim 2 wherein n is
 0. 8. The method of claim 7wherein X₄ is benzoyl, acetyl or levulinyl.
 9. The method of claim 8wherein A is phenyl, with —OX₄ attached at the ortho or para position.10. The method of claim 8 wherein —OX₄ is in the ortho position.
 11. Themethod of claim 10 wherein at least one of X₁ and X₅ is O.
 12. Themethod of claim 10 wherein of X₁ and X₅ are each O.
 13. The method ofclaim 10 wherein at least one of X₁ and X₅ is S.
 14. The method of claim8 wherein each R₆ is isopropyl.
 15. The method of claim 1 wherein n is0; A is phenyl with —OX₄ attached at the ortho or para position, X₄ isacetyl, benzoyl or levulinyl; X₁ and X₅ are each O; and R₅ isdiisopropylamino.
 16. The method of claim 15 wherein —OX₄ is acetyl inthe ortho position.
 17. The method of claim 1 wherein the compound ofFormula II is obtained by reaction of a compound having Formula V:

with a compound having the Formula VI:

in the presence of an acid.
 18. The method of claim 1 wherein thecompound of Formula II is obtained by the steps of: (a) reacting acompound having Formula V:

with a chlorophosphine compund of formula ClP(R₅)₂ in the presence of abase; and (b) contacting the product if step (a) with a compound ofFormula XX:

in the presence of an acid.
 19. The method of claim 18 wherein R₅ isdiisopropylamino.
 20. A compound having Formula VII:

wherein: D is a monocyclic or bicyclic aromatic ring system; R₁₁ and R₁₂are each independently H, alkyl, aryl, heteroalkyl, heteroaryl, alkaryl,or aralkyl; or R₁₁ and R₁₂ together with the carbon atoms to which theyare attached form an optionally substituted aliphatic or aromatic ringhaving from 4 to 6 ring atoms; X₄ is alkaryl, aralkyl, sulfoxyl,sulfonyl, thio, substituted sulfoxyl, substituted sulfonyl, orsubstituted thio, wherein said substituent is alkyl, aryl, or alkaryl;or X₄ is a group of formula —C(═O)—(O)_(aa)—R₄₀ where aa is 0 or 1 andR₄₀ is lower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl,aryl, aralkyl or heteroaryl groups are optionally substituted with oneor more alkyl, aryl, aralkyl, halo or acetyl groups; or X₄ is a group offormula —(—CH₂—CH₂—)_(d)Si(R₉)₃ where d is 0 or 1; each R₉ is,independently, alkyl having 1 to about 10 carbon atoms, or aryl having 6to about 10 carbon atoms; X₁ and X₅ are each independently O or S; A is(R₇)(R₈)P— or (R₇)(R₈)P(═X₂)—; X₂ is O or S; R₈ is R₅, or has theFormula VIII:

 wherein each R₁, is, independently, H, hydroxyl, C₁-C₂₀ alkyl, C₃-C₂₀alkenyl, C₂-C₂₀ alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso,nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, or polyether; or R₁ is agroup of formula Z—R₂₂—(R₂₃)_(v); Z is O, S, NH, or N—R₂₂—(R₂₃)_(v); R₂₂is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl; R₂₃ is hydrogen,amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso,nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, polyether, a group thatenhances the pharmacodynamic properties of oligonucleotides, or a groupthat enhances the pharmacokinetic properties of oligonucleotides; v isfrom 0 to about 10; or R₁ has the formula:

y1 is 0 or 1; y2 is independently 0 to 10; y3 is 1 to 10; E is C₁-C₁₀alkyl, N(Q₁) (Q₂) or N═C(Q₁) (Q₂); each Q₁ and Q₂ is, independently, H,C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, dialkylaminoalkyl, a nitrogenprotecting group, a tethered or untethered conjugate group, a linker toa solid support; or Q₁ and Q₂, together, are joined in a nitrogenprotecting group or a ring structure that can include at least oneadditional heteroatom selected from N and O; or R₁ has one of formula XIor XII:

 wherein Z₀ is O, S, or NH; q¹ is from 0 to 10; q² is from 1 to 10; q³is 0 or 1; q⁴ is, 0, 1 or 2; Z₄ is OM₁, SM₁, or N(M₁)₂; each M₁ is,independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C(═NH)N(H)M₂,C(═O)N(H)M₂ or OC(═O)N(H)M₂; M₂ is H or C₁-C₈ alkyl; Z₁, Z₂ and Z₃comprise a ring system having from about 4 to about 7 carbon atoms, orhaving from about 3 to about 6 carbon atoms and 1 or 2 hetero atomswherein said hetero atoms are selected from oxygen, nitrogen and sulfur,and wherein said ring system is aliphatic, unsaturated aliphatic,aromatic, or saturated or unsaturated heterocyclic; and Z₅ is alkyl orhaloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having6 to about 14 carbon atoms, N(Q₁) (Q₂), OQ₁, halo, SQ₁ or CN; R₅ is—N(R₆)₂, or a heterocycloalkyl or heterocycloalkenyl ring containingfrom 4 to 7 atoms, and having up to 3 heteroatoms selected fromnitrogen, sulfur, and oxygen; and R₆ is straight or branched chain alkylhaving from 1 to 10 carbons; each B, independently, is a naturallyoccurring or non-naturally occurring nucleobase or a protected naturallyoccurring or non-naturally occurring nucleobase; R₂ is hydrogen, ahydroxyl protecting group, or a linker connected to a solid support; Mis an optionally protected internucleoside linkages; m is 0 to about 50;and R₇ is R₅, or has the Formula IX:

 wherein: R₃ is hydrogen, a hydroxyl protecting group, or a linkerconnected to a solid support; n is 0 to about 50; with the proviso thatthe sum of m and n do not exceed
 50. 21. The compound of claim 20wherein A is phenyl or naphthalene.
 22. The compound of claim 20 whereinX₄ is benzoyl, acetyl or levulinyl.
 23. The compound of claim 21 whereinX₄ is benzoyl, acetyl or levulinyl.
 24. The compound of claim 23 whereinA is phenyl, with —OX₄ in the ortho or para position.
 25. The compoundof claim 24 wherein —OX₄ is in the ortho position.
 26. The compound ofclaim 20 wherein at least one of X₁ and X₅ is O.
 27. The compound ofclaim 20 wherein X₁ and X₅ are each O.
 28. The compound of claim 20wherein R₁₁ and R₁₂ are each H; A is phenyl with —OX₄ in the ortho orpara position, or A is naphthalene connected to X₅ at the 1-position,with the moiety —OX₄ being at the 5- or 6-position; X₄ is benzoyl,acetyl or levulinyl; and X₁ and X₅ are each O.
 29. The compound of claim27 wherein X₄ is acetyl; and A is phenyl with —OX₄ in the orthoposition.
 30. The compound of claim 20 wherein R₈ is R₅ and R₇ has theFormula IX.
 31. The compound of claim 30 wherein n is
 0. 32. Thecompound of claim 28 wherein R₈ is R₅ and R₇ has the Formula IX.
 33. Thecompound of claim 32 wherein n is
 0. 34. The compound of claim 33wherein A is phenyl with —OX₄ in the ortho or para position.
 35. Thecompound of claim 34 wherein X₄ is acetyl.
 36. The compound of claim 35wherein R₅ is diisopropylamino.
 37. The compound of claim 20 wherein R₈has the Formula VIII, and R₇ has the Formula IX.
 38. The compound ofclaim 37 wherein n is
 0. 39. The compound of claim 37 wherein m is 0.40. The compound of claim 37 wherein R₁₁ and R₁₂ are each H; A is phenylwith —OX₄ in the ortho or para position, or A is naphthalene connectedto X₅ at the 1-position, with the moiety —OX₄ being at the 5- or6-position; X₄ is benzoyl, acetyl or levulinyl; and X₁ and X₅ are eachO.
 41. The compound of claim 20 wherein D is (R₇) (R₈)P—.
 42. A compoundcomprising a moiety having the Formula:

wherein: A is a monocyclic or bicyclic aromatic ring system; R₁₁ and R₁₂are each independently H, alkyl, aryl, heteroalkyl, heteroaryl, alkaryl,or aralkyl; or R₁₁ and R₁₂ together with the carbon atoms to which theyare attached form an optionally substituted aliphatic or aromatic ringhaving from 4 to 6 ring atoms; X₄ is alkaryl, aralkyl, sulfoxyl,sulfonyl, thio, substituted sulfoxyl, substituted sulfonyl, orsubstituted thio, wherein said substituent is alkyl, aryl, or alkaryl;or X₄ is a group of formula —C(═O)—(O)_(aa)—R₄₀ where aa is 0 or 1 andR₄₀ is lower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl,aryl, aralkyl or heteroaryl groups are optionally substituted with oneor more alkyl, aryl, aralkyl, halo or acetyl groups; or X₄ is a group offormula —(—CH₂—CH₂—)_(d)Si(R₉)₃ where d is 0 or 1; each R₉ is,independently, alkyl having 1 to about 10 carbon atoms, or aryl having 6to about 10 carbon atoms; and X₁ and X₅ are each independently O or S.43. The compound of claim 42 wherein A is phenyl with the moiety —OX₄ inthe ortho or para position.
 44. The compound of claim 43 wherein themoiety —OX₄ is in the ortho position.
 45. The compound of claim 42wherein A is phenyl with the moiety —OX₄ in the ortho or para position;X₄ is acetyl, benzoyl, or levulinyl; X₁ and X₅ are each O; and R₁₁ andR₁₂ are each H.
 46. A compound having Formula:

wherein: A is a monocyclic or bicyclic aromatic ring system; R₁₁ and R₁₂are each independently H, alkyl, aryl, heteroalkyl, heteroaryl, alkaryl,or aralkyl; or R₁₁ and R₁₂ together with the carbon atoms to which theyare attached form an optionally substituted aliphatic or aromatic ringhaving from 4 to 6 ring atoms; X₄ is alkaryl, aralkyl, sulfoxyl,sulfonyl, thio, substituted sulfoxyl, substituted sulfonyl, orsubstituted thio, wherein said substituent is alkyl, aryl, or alkaryl;or X₄ is a group of formula —C(═O)—(O)_(aa)—R₄₀ where aa is 0 or 1 andR₄₀ is lower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl,aryl, aralkyl or heteroaryl groups are optionally substituted with oneor more alkyl, aryl, aralkyl, halo or acetyl groups; or X₄ is a group offormula —(—CH₂—CH₂—)_(d)Si(R₉)₃ where d is 0 or 1; each R₉ is,independently, alkyl having 1 to about 10 carbon atoms, or aryl having 6to about 10 carbon atoms; X₁ is O or S; X₂ is O or S; each R₁, is,independently, H, hydroxyl, C₁-C₂₀ alkyl, C₃-C₂₀ alkenyl, C₂-C₂₀alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso, nitrile,trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, or polyether; or R₁ is agroup of formula Z—R₂₂—(R₂₃)_(v); Z is O, S, NH, or N—R₂₂—(R₂₃)_(v); R₂₂is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl; R₂₃ is hydrogen,amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso,nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole. azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, polyether, a group thatenhances the pharmacodynamic properties of oligonucleotides, or a groupthat enhances the pharmacokinetic properties of oligonucleotides; v isfrom 0 to about 10; or R₁ has the formula:

y1 is 0 or 1; y2 is independently 0 to 10; y3 is 1 to 10; E is C₁-C₁₀alkyl, N(Q₁) (Q₂) or N═C(Q₁) (Q₂); each Q₁ and Q₂ is, independently, H,C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, dialkylaminoalkyl, a nitrogenprotecting group, a tethered or untethered conjugate group, a linker toa solid support; or Q₁ and Q₂, together, are joined in a nitrogenprotecting group or a ring structure that can include at least oneadditional heteroatom selected from N and O; or R₁ has one of formula XIor XII:

 wherein Z₀ is O, S, or NH; q¹ is from 0 to 10; q² is from 1 to 10; q³is 0 or 1; q⁴ is, 0, 1 or 2; Z₄ is OM₁, SM₁, or N(M₁)₂; each M₁ is,independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C(═NH)N(H)M₂,C(═O)N(H)M₂ or OC(═O)N(H)M₂; M₂ is H or C₁-C₈ alkyl; Z₁, Z₂ and Z₃comprise a ring system having from about 4 to about 7 carbon atoms, orhaving from about 3 to about 6 carbon atoms and 1 or 2 hetero atomswherein said hetero atoms are selected from oxygen, nitrogen and sulfur,and wherein said ring system is aliphatic, unsaturated aliphatic,aromatic, or saturated or unsaturated heterocyclic; and Z₅ is alkyl orhaloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having6 to about 14 carbon atoms, N(Q₁) (Q₂), OQ₁, halo, SQ₁ or CN; R₂ ishydrogen, a hydroxyl protecting group, or a linker connected to a solidsupport; R₃ is hydrogen, a hydroxyl protecting group, or a linkerconnected to a solid support, provided that R₂ and R₃ are not bothsimultaneously a linker connected to a solid support; M is aninternucleoside linkage; m and n are each independently from 0 to about50, provided that the sum of m and n does not exceed 50; and each B,independently, is a naturally occurring or non-naturally occurringnucleobase or a protected naturally occurring or non-naturally occurringnucleobase.
 47. The compound of claim 46 wherein A is phenyl with themoiety —OX₄ in the ortho or para position, or A is naphthalene connectedto X₅ at the 1-position, with the moiety —OX₄ being in the 5- or6-position.
 48. The compound of claim 47 wherein R₂ is a linkerconnected to a solid support.
 49. The compound of claim 48 wherein n is0.
 50. A method for the preparation of a compound of Formula II:

wherein: A is a monocyclic or bicyclic aromatic ring system; R₁₁ and R₁₂are each independently H, alkyl, aryl, heteroalkyl, heteroaryl, alkaryl,or aralkyl; or R₁₁ and R₁₂ together with the carbon atoms to which theyare attached form an optionally substituted aliphatic or aromatic ringhaving from 4 to 6 ring atoms; X₄ is alkaryl, aralkyl, sulfoxyl,sulfonyl, thio, substituted sulfoxyl, substituted sulfonyl, orsubstituted thio, wherein said substituent is alkyl, aryl, or alkaryl;or X₄ is a group of formula —C(═O)—(O)_(aa)—R₄₀ where aa is 0 or 1 andR₄₀ is lower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl,aryl, aralkyl or heteroaryl groups are optionally substituted with oneor more alkyl, aryl, aralkyl, halo or acetyl groups; or X₄ is a group offormula —(—CH₂—CH₂—)_(d)Si(R₉)₃ where d is 0 or 1; each R₉ is,independently, alkyl having 1 to about 10 carbon atoms, or aryl having 6to about 10 carbon atoms; X₁ and X₅ are each independently O or S; eachR₁, is, independently, H, hydroxyl, C₁-C₂₀ alkyl, C₃-C₂₀ alkenyl, C₂-C₂₀alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso, nitrile,trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, or polyether; or R₁ is agroup of formula Z—R₂₂—(R₂₃)_(v); Z is O, S, NH, or N—R₂₂—(R₂₃)_(v); R₂₂is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl; R₂₃ is hydrogen,amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso,nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, polyether, a group thatenhances the pharmacodynamic properties of oligonucleotides, or a groupthat enhances the pharmacokinetic properties of oligonucleotides; v isfrom 0 to about 10; or R₁ has the formula:

y1 is 0 or 1; y2 is independently 0 to 10; y3 is 1 to 10; E is C₁-C₁₀alkyl, N(Q₁) (Q₂) or N═C(Q₁) (Q₂); each Q₁ and Q₂ is, independently, H,C₁-C₁₀alkyl, substituted C₁-C₁₀ alkyl, dialkylaminoalkyl, a nitrogenprotecting group, a tethered or untethered conjugate group, a linker toa solid support; or Q₁ and Q₂, together, are joined in a nitrogenprotecting group or a ring structure that can include at least oneadditional heteroatom selected from N and O; or R₁ has one of formula XIor XII:

 wherein Z₀ is O, S, or NH; q¹ is from 0 to 10; q² is from 1 to 10; q³is 0 or 1; q⁴ is, 0, 1 or 2; Z₄ is OM₁, SM₁, or N(M₁)₂; each M₁ is,independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C(═NH)N(H)M₂,C(═O)N(H)M₂ or OC(═O)N(H)M₂; M₂ is H or C₁-C₈ alkyl; Z₁, Z₂ and Z₃comprise a ring system having from about 4 to about 7 carbon atoms, orhaving from about 3 to about 6 carbon atoms and 1 or 2 hetero atomswherein said hetero atoms are selected from oxygen, nitrogen and sulfur,and wherein said ring system is aliphatic, unsaturated aliphatic,aromatic, or saturated or unsaturated heterocyclic; and Z₅ is alkyl orhaloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having6 to about 14 carbon atoms, N(Q₁) (Q₂), OQ₁, halo, SQ₁ or CN; R₃ ishydrogen, a hydroxyl protecting group, or a linker connected to a solidsupport; each B, independently, is a naturally occurring ornon-naturally occurring nucleobase or a protected naturally occurring ornon-naturally occurring nucleobase; n is 0 to about 50; M is anoptionally protected internucleoside linkage; R₅ is —N(R₆)₂, or aheterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur,and oxygen; and R₆ is straight or branched chain alkyl having from 1 to10 carbons; comprising: (a) selecting a 5′-protected nucleoside havingFormula V:

 and (b) reacting the nucleoside with a compound having the Formula VI:

in the presence of an acid.
 51. A method for the preparation of acompound of Formula II:

wherein: A is a monocyclic or bicyclic aromatic ring system; R₁₁ and R₁₂are each independently H, alkyl, aryl, heteroalkyl, heteroaryl, alkaryl,or aralkyl; or R₁₁ and R₁₂ together with the carbon atoms to which theyare attached form an optionally substituted aliphatic or aromatic ringhaving from 4 to 6 ring atoms; X₄ is alkaryl, aralkyl, sulfoxyl,sulfonyl, thio, substituted sulfoxyl, substituted sulfonyl, orsubstituted thio, wherein said substituent is alkyl, aryl, or alkaryl;or X₄ is a group of formula —C(═O)—(O)_(aa)—R₄₀ where aa is 0 or 1 andR₄₀ is lower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl,aryl, aralkyl or heteroaryl groups are optionally substituted with oneor more alkyl, aryl, aralkyl, halo or acetyl groups; or X₄ is a group offormula —(—CH₂—CH₂—)_(d)Si(R₉)₃ where d is 0 or 1; each R₉ is,independently, alkyl having 1 to about 10 carbon atoms, or aryl having 6to about 10 carbon atoms; X₁ and X₅ are each independently O or S; eachR₁, is, independently, H, hydroxyl, C₁-C₂₀ alkyl, C₃-C₂₀ alkenyl, C₂-C₂₀alkynyl, halogen, thiol, keto, carboxyl, nitro, nitroso, nitrile,trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, or polyether; or R₁ is agroup of formula Z—R₂₂—(R₂₃)_(v); Z is O, S, NH, or N—R₂₂—(R₂₃)_(v); R₂₂is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl; R₂₃ is hydrogen,amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso,nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl,N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl,amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino,isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl,heterocycle, carbocycle, intercalator, reporter molecule, conjugate,polyamine, polyamide, polyalkylene glycol, polyether, a group thatenhances the pharmacodynamic properties of oligonucleotides, or a groupthat enhances the pharmacokinetic properties of oligonucleotides; v isfrom 0 to about 10; or R₁ has the formula:

y1 is 0 or 1; y2 is independently 0 to 10; y3 is 1 to 10; E is C₁-C₁₀alkyl, N(Q₁) (Q₂) or N═C(Q₁) (Q₂); each Q₁ and Q₂ is, independently, H,C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, dialkylaminoalkyl, a nitrogenprotecting group, a tethered or untethered conjugate group, a linker toa solid support; or Q₁ and Q₂, together, are joined in a nitrogenprotecting group or a ring structure that can include at least oneadditional heteroatom selected from N and O; or R₁ has one of formula XIor XII:

 wherein Z₀ is O, S, or NH; q¹ is from 0 to 10; q² is from 1 to 10; q³is 0 or 1; q⁴ is, 0, 1 or 2; Z₄ is OM₁, SM₁, or N(M₁)₂; each M₁ is,independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C(═NH)N(H)M₂,C(═O)N(H)M₂ or OC(═O)N(H)M₂; M₂ is H or C₁-C₈ alkyl; Z₁, Z₂ and Z₃comprise a ring system having from about 4 to about 7 carbon atoms, orhaving from about 3 to about 6 carbon atoms and 1 or 2 hetero atomswherein said hetero atoms are selected from oxygen, nitrogen and sulfur,and wherein said ring system is aliphatic, unsaturated aliphatic,aromatic, or saturated or unsaturated heterocyclic; and Z₅ is alkyl orhaloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having6 to about 14 carbon atoms, N(Q₁) (Q₂), OQ₁, halo, SQ₁ or CN; R₃ ishydrogen, a hydroxyl protecting group, or a linker connected to a solidsupport; each B, independently, is a naturally occurring ornon-naturally occurring nucleobase or a protected naturally occurring ornon-naturally occurring nucleobase; n is 0 to about 50; M is anoptionally protected internucleoside linkage; R₅ is —N(R₆)₂, or aheterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur,and oxygen; and R₆ is straight or branched chain alkyl having from 1 to10 carbons; comprising: (a) selecting a 5′-protected nucleoside ofFormula V:

 wherein R₃ is a hydroxyl protecting group; (b) reacting the protectednucleoside with a chlorophosphine compund of formula ClP(R₅)₂ in thepresence of a base; and (c) contacting the product of step (b) with acompound of Formula XX:

in the presence of an acid; to form the nucleoside phosphoramidite. 52.A compound having the formula:

wherein A is a monocyclic or bicyclic aromatic ring system; R₁₁ and R₁₂are each H, independently alkyl, aryl, heteroalkyl, heteroaryl, alkaryl,or aralkyl; or R₁₁ and R₁₂ together with the carbon atoms to which theyare attached form an optionally substituted aliphatic or aromatic ringhaving from 4 to 6 ring atoms; X₄ is alkaryl, aralkyl, sulfoxyl,sulfonyl, thio, substituted sulfoxyl, substituted sulfonyl, orsubstituted thio, wherein said substituent is alkyl, aryl, or alkaryl;or X₄ is a group of formula —C(═O)—(O)_(aa)—R₄₀ where aa is 0 or 1 andR₄₀ is lower alkyl, aryl, aralkyl, heteroaryl wherein said lower alkyl,aryl, aralkyl or heteroaryl groups are optionally substituted with oneor more alkyl, aryl, aralkyl, halo or acetyl groups; or X₄ is a group offormula —(—CH₂—CH₂—)_(d)Si(R₉)₃ where d is 0 or 1; each R₉ is,independently, alkyl having 1 to about 10 carbon atoms, or aryl having 6to about 10 carbon atoms; X₁ and X₅ are each independently O or S; R₅ is—N(R₆)₂, or a heterocycloalkyl or heterocycloalkenyl ring containingfrom 4 to 7 atoms, and having up to 3 heteroatoms selected fromnitrogen, sulfur, and oxygen; and R₆ is straight or branched chain alkylhaving from 1 to 10 carbons.
 53. The compound of claim 52 wherein A isphenyl with the moiety —OX₄ in the ortho or para position, or A isnaphthalene connected to X₅ at the 1-position, with the moiety —OX₄being in the 5- or 6-position, and X₁ and X₅ are each O.
 54. Thecompound of claim 53 wherein X₄ is benzoyl, acetyl, or levulinyl. 55.The compound of claim 54 wherein X₃ is —N(R₆)₂ where R₆ is isopropyl.56. The compound of claim 55 wherein X₂ is chlorine.
 57. The method ofclaim 1 further comprising iterative repetition of steps (a) and (b) toproduce an oligomeric compound having from 4 to about 50 nucleobases.